Protease activity as a prognostic factor for wound healing in venous leg ulcers.

BACKGROUND: Venous leg ulcers (VLUs) are a common type of complex wound that have a negative impact on people's lives and incur high costs for health services and society. It has been suggested that prolonged high levels of protease activity in the later stages of the healing of chronic wounds may be associated with delayed healing. Protease modulating treatments have been developed which seek to modulate protease activity and thereby promote healing in chronic wounds. OBJECTIVES: To determine whether protease activity is an independent prognostic factor for the healing of venous leg ulcers. SEARCH METHODS: In February 2018, we searched the following databases: Cochrane Central Register of Controlled Trials (CENTRAL), Ovid MEDLINE, Ovid Embase and CINAHL. SELECTION CRITERIA: We included prospective and retrospective longitudinal studies with any follow-up period that recruited people with VLUs and investigated whether protease activity in wound fluid was associated with future healing of VLUs. We included randomised controlled trials (RCTs) analysed as cohort studies, provided interventions were taken into account in the analysis, and case-control studies if there were no available cohort studies. We also included prediction model studies provided they reported separately associations of individual prognostic factors (protease activity) with healing. Studies of any type of protease or combination of proteases were eligible, including proteases from bacteria, and the prognostic factor could be examined as a continuous or categorical variable; any cut-off point was permitted. The primary outcomes were time to healing (survival analysis) and the proportion of people with ulcers completely healed; the secondary outcome was change in ulcer size/rate of wound closure. We extracted unadjusted (simple) and adjusted (multivariable) associations between the prognostic factor and healing. DATA COLLECTION AND ANALYSIS: Two review authors independently assessed studies for inclusion at each stage, and undertook data extraction, assessment of risk of bias and GRADE assessment. We collected association statistics where available. No study reported adjusted analyses: instead we collected unadjusted results or calculated association measures from raw data. We calculated risk ratios when both outcome and prognostic factor were dichotomous variables. When the prognostic factor was reported as continuous data and healing outcomes were dichotomous, we either performed regression analysis or analysed the impact of healing on protease levels, analysing as the standardised mean difference. When both prognostic factor and outcome were continuous data, we reported correlation coefficients or calculated them from individual participant data.We displayed all results on forest plots to give an overall visual representation. We planned to conduct meta-analyses where this was appropriate, otherwise we summarised narratively. MAIN RESULTS: We included 19 studies comprising 21 cohorts involving 646 participants. Only 11 studies (13 cohorts, 522 participants) had data available for analysis. Of these, five were prospective cohort studies, four were RCTs and two had a type of case-control design. Follow-up time ranged from four to 36 weeks. Studies covered 10 different matrix metalloproteases (MMPs) and two serine proteases (human neutrophil elastase and urokinase-type plasminogen activators). Two studies recorded complete healing as an outcome; other studies recorded partial healing measures. There was clinical and methodological heterogeneity across studies; for example, in the definition of healing, the type of protease and its measurement, the distribution of active and bound protease species, the types of treatment and the reporting of results. Therefore, meta-analysis was not performed. No study had conducted multivariable analyses and all included evidence was of very low certainty because of the lack of adjustment for confounders, the high risk of bias for all studies except one, imprecision around the measures of association and inconsistency in the direction of association. Collectively the research indicated complete uncertainty as to the association between protease activity and VLU healing. AUTHORS' CONCLUSIONS: This review identified very low validity evidence regarding any association between protease activity and VLU healing and there is complete uncertainty regarding the relationship. The review offers information for both future research and systematic review methodology.

Protease-modulating matrix treatments for healing venous leg ulcers.

BACKGROUND: Venous leg ulcers (VLUs) are open skin wounds on the lower leg that occur because of poor blood flow in the veins of the leg; leg ulcers can last from weeks to years, and are both painful and costly. Prevalence in the UK is about 2.9 cases per 10,000 people. First-line treatment for VLUs is compression therapy, but around 60% of people have unhealed ulcers after 12 weeks' treatment and about 40% after 24 weeks; therefore, there is scope for further improvement. Limited evidence suggests non-healing leg ulcers may have persisting elevated levels of proteases, which is thought to deter the later stages of healing; thus, timely protease-modulating matrix (PMM) treatments may improve healing by physically removing proteases from the wound fluid. OBJECTIVES: To determine the effects of protease-modulating matrix (PMM) treatments on the healing of venous leg ulcers, in people managed in any care setting. SEARCH METHODS: In September 2016 we searched: the Cochrane Wounds Specialised Register; CENTRAL; Ovid MEDLINE; Ovid MEDLINE (In-Process & Other Non-Indexed Citations); Ovid Embase and EBSCO CINAHL Plus. We also searched clinical trials registries for ongoing and unpublished studies, and scanned reference lists of relevant included studies as well as reviews, meta-analyses and health technology reports to identify additional studies. There were no restrictions with respect to language, date of publication or study setting. SELECTION CRITERIA: We searched for published or unpublished randomised controlled trials (RCTs) that evaluated PMM treatments for VLUs. We defined PMM treatments as those with a purposeful intent of reducing proteases. Wound healing was the primary endpoint. DATA COLLECTION AND ANALYSIS: Two review authors independently performed study selection, risk of bias assessment and data extraction. MAIN RESULTS: We included 12 studies (784 participants) in this review; sample sizes ranged from 10 to 187 participants (median 56.5). One study had three arms that were all relevant to this review and all the other studies had two arms. One study was a within-participant comparison. All studies were industry funded. Two studies provided unpublished data for healing.Nine of the included studies compared PMM treatments with other treatments and reported results for the primary outcomes. All treatments were dressings. All studies also gave the participants compression bandaging. Seven of these studies were in participants described as having 'non-responsive' or 'hard-to-heal' ulcers. Results, reported at short, medium and long durations and as time-to-event data, are summarised for the comparison of any dressing regimen incorporating PMM versus any other dressing regimen. The majority of the evidence was of low or very low certainty, and was mainly downgraded for risk of bias and imprecision.It is uncertain whether PMM dressing regimens heal VLUs quicker than non-PMM dressing regimens (low-certainty evidence from 1 trial with 100 participants) (HR 1.21, 95% CI 0.74 to 1.97).In the short term (four to eight weeks) it is unclear whether there is a difference between PMM dressing regimens and non-PMM dressing regimens in the probability of healing (very low-certainty evidence, 2 trials involving 207 participants).In the medium term (12 weeks), it is unclear whether PMM dressing regimens increase the probability of healing compared with non-PMM dressing regimens (low-certainty evidence from 4 trials with 192 participants) (RR 1.28, 95% CI 0.95 to 1.71). Over the longer term (6 months), it is also unclear whether there is a difference between PMM dressing regimens and non-PMM dressing regimens in the probability of healing (low certainty evidence, 1 trial, 100 participants) (RR 1.06, 95% CI 0.80 to 1.41).It is uncertain whether there is a difference in adverse events between PMM dressing regimens and non-PMM dressing regimens (low-certainty evidence from 5 trials, 363 participants) (RR 1.03, 95% CI 0.75 to 1.42). It is also unclear whether resource use is lower for PMM dressing regimens (low-certainty evidence, 1 trial involving 73 participants), or whether mean total costs in a German healthcare setting are different (low-certainty evidence, 1 trial in 187 participants). One cost-effectiveness analysis was not included because effectiveness was not based on complete healing. AUTHORS' CONCLUSIONS: The evidence is generally of low certainty, particularly because of risk of bias and imprecision of effects. Within these limitations, we are unclear whether PMM dressing regimens influence venous ulcer healing relative to dressing regimens without PMM activity. It is also unclear whether there is a difference in rates of adverse events between PMM and non-PMM treatments. It is uncertain whether either resource use (products and staff time) or total costs associated with PMM dressing regimens are different from those for non-PMM dressing regimens. More research is needed to clarify the impact of PMM treatments on venous ulcer healing.

Defining a new diagnostic assessment parameter for wound care: Elevated protease activity, an indicator of nonhealing, for targeted protease-modulating treatment.

It is widely accepted that elevated protease activity (EPA) in chronic wounds impedes healing. However, little progress has occurred in quantifying the level of protease activity that is detrimental for healing. The aim of this study was to determine the relationship between inflammatory protease activity and wound healing status, and to establish the level of EPA above which human neutrophil-derived elastase (HNE) and matrix metalloproteases (MMP) activities correlate with nonhealing wounds. Chronic wound swab samples (n = 290) were collected from four wound centers across the USA to measure HNE and MMP activity. Healing status was determined according to percentage reduction in wound area over the previous 2-4 weeks; this was available for 211 wounds. Association between protease activity and nonhealing wounds was determined by receiver operating characteristic analysis (ROC), a statistical technique used for visualizing and analyzing the performance of diagnostic tests. ROC analysis showed that area under the curve (AUC) for HNE were 0.69 for all wounds and 0.78 for wounds with the most reliable wound trajectory information, respectively. For MMP, the corresponding AUC values were 0.70 and 0.82. Analysis suggested that chronic wounds having values of HNE >5 and/or MMP ≥13, should be considered wound healing impaired. EPA is indicative of nonhealing wounds. Use of a diagnostic test to detect EPA in clinical practice could enable clinicians to identify wounds that are nonhealing, thus enabling targeted treatment with protease modulating therapies.

A 'test and treat' strategy for elevated wound protease activity for healing in venous leg ulcers.

BACKGROUND: Venous leg ulcers are a common and recurring type of complex wound. They can be painful, malodorous, prone to infection and slow to heal. Standard treatment includes compression therapy and a dressing. The use of protease-modulating treatments for venous leg ulcers is increasing. These treatments are based on some evidence that a proportion of slow to heal ulcers have elevated protease activity in the wound. Point-of-care tests which aim to detect elevated protease activity are now available. A 'test and treat' strategy involves testing for elevated proteases and then using protease-modulating treatments in ulcers which show elevated protease levels. OBJECTIVES: To determine the effects on venous leg ulcer healing of a 'test and treat' strategy involving detection of high levels of wound protease activity and treatment with protease-modulating therapies, compared with alternative treatment strategies such as using the same treatment for all participants or using a different method of treatment selection. SEARCH METHODS: We searched the following electronic databases to identify reports of relevant randomised clinical trials: The Cochrane Wounds Group Specialised Register (January 2016), the Cochrane Central Register of Controlled Trials (CENTRAL, The Cochrane Library) Issue 12, 2015); Ovid MEDLINE (1946 to January 2016); Ovid MEDLINE (In-Process & Other Non-Indexed Citations January 2016); Ovid EMBASE (1974 to January 2016); EBSCO CINAHL (1937 to January 2016). We also searched three clinical trials registers, reference lists and the websites of regulatory agencies. There were no restrictions with respect to language, date of publication or study setting. SELECTION CRITERIA: Published or unpublished RCTs which assessed a test and treat strategy for elevated protease activity in venous leg ulcers in adults compared with an alternative treatment strategy. The test and treat strategy needed to be the only systematic difference between the groups. DATA COLLECTION AND ANALYSIS: Two review authors independently performed study selection; we planned that two authors would also assess risk of bias and extract data. MAIN RESULTS: We did not identify any studies which met the inclusion criteria for this review. We identified one ongoing study; it was unclear whether this would be eligible for inclusion. AUTHORS' CONCLUSIONS: Currently there is no randomised evidence on the impact of a test and treat policy for protease levels on outcomes in people with venous leg ulcers.

Role of matrix metalloproteinases in non-healing venous ulcers.

Chronic venous ulceration (CVU) of the lower limbs is a common condition affecting 1% of the adult population in Western countries, which is burdened with a high complication rate and a marked reduction in the quality of life often due to prolonged healing time. Several metalloproteinases (MMPs) such as MMP-9 together with neutrophil gelatinase-associated lipocalin (NGAL) appear to be involved in the onset and healing phases of venous ulcer, but it is still unclear how many biochemical components are responsible for prolonged healing time in those ulcers. In this study, we evaluate the role of MMP-1 and MMP-8 in long lasting and refractory venous ulcers. In a 2-year period we enroled 45 patients (28 female and 17 male, median age 65) with CVU. The enroled population was divided into two groups: group I were patients with non-healing ulcers (ulcers that had failed to heal for more than 2 months despite appropriate treatments) and group II were patients with healing ulcers (ulcers in healing phases). MMP-1 and MMP-8 were measured in fluids and tissues of healing and non-healing ulcers by means of enzyme-linked immunosorbent assay (ELISA) and Western blot analysis, respectively. In particular the patterns of the collagenases MMP-1 and MMP-8 in healing wounds were distinct, with MMP-8 appearing in significantly greater amounts especially in the non-healing group. Our findings suggest that MMP-1, and MMP-8 are overexpressed in long lasting CVU. Therefore, this dysregulation may represent the main cause of the pathogenesis of non-healing CVU.

The direct inhibition of MMP-2 and MMP-9 by an enzyme alginogel: a possible mechanism of healing support for venous leg ulcers.

Flaminal Forte is an enzyme alginogel,whose activity depends on the absorption and binding of matrix metalloproteinases (MMPs), which are known to play a crucial role in delayed wound healing. The aim of the study was to evaluate the influence of Flaminal on MMP-2/-9 activity in ulcer exudate, ex vivo. Eight patients with bilateral venous leg ulcers were treated for 4 weeks with Flaminal Forte covered by hydrocolloid ('F' wounds), or with hydrocolloid alone ('H' wounds) as a reference control. Clinical assessment did not reveal any differences between F and H wounds regarding surface reduction and general wound condition. Nevertheless, although non-significant, there was a visible difference in peri-wound skin appearance in F wounds, as compared to H wounds. The wound exudate contained high MMP-2/-9 levels, which gradually decreased as wounds healed. The attenuation of MMPs was stronger in F than in H exudate, however, in standard zymography this difference appeared non-significant. Real-time zymography revealed that Flaminal mediated a powerful direct inhibition of gelatinolytic activity of wound exudate and of recombinant MMP-2/-9 in vitro.

Chronic venous leg ulcers are associated with high levels of metalloproteinases-9 and neutrophil gelatinase-associated lipocalin.

Venous ulcers are related to dysfunctions in extracellular matrix. Both matrix metalloproteinases (MMP) and neutrophil gelatinase-associated lipocalin (NGAL) could play a role in the healing process in patients with chronic venous ulcers. We evaluated the role of MMP-9 and NGAL in the healing process in venous ulceration. We performed an open-label, parallel groups, single clinical center study. Patients with chronic venous leg ulcers represented the test group (Group I), whereas patients without chronic ulcers represented the control group (Group II). In Group I plasma and wound fluid samples were collected at the time of admission, at the time of the surgery, and at the follow-up, while ulcer tissues were taken at the time of the surgery. In Group II, plasma and wound fluid were collected at admission and at the time of the surgery, whereas skin tissues were collected at the time of the surgery. Enzyme-linked immunosorbent assay test was used to evaluate the levels of MMP-9 and NGAL in plasma and wound fluid, whereas Western blot analysis was performed to estimate the expression of MMP-9 and NGAL in tissues. Enzyme-linked immunosorbent assay tests revealed significantly higher levels of MMP-9 and NGAL in both plasma and wound fluid of patients with ulcers compared to patients without ulcers (p < 0.01). Moreover, Western blot analysis documented an increased expression of MMP-9 and NGAL in biopsy tissue of patients with ulcers compared to patients without ulcers (p < 0.01). In conclusion MMP-9 and NGAL may correlate with the clinical course of venous ulcers.

Wound debridement potential of glycosidases of the wound-healing maggot, Lucilia sericata.

The wound-healing maggot, Lucilia sericata Meigen (Diptera: Calliphoridae), degrades extracellular matrix components by releasing enzymes. The purpose of this study was to investigate the glycosylation profiles of wound slough/eschar from chronic venous leg ulcers and the complementary presence of glycosidase activities in first-instar excretions/secretions (ES1) and to define their specificities. The predominant carbohydrate moieties present in wound slough/eschar were determined by probing one-dimensional Western blots with conjugated lectins of known specificities. The presence of specific glycosidase activities in ES1 was determined using chromogenic and fluorogenic substrates. The removal of carbohydrate moieties from slough/eschar proteins by glycosidases in ES1 was determined by two-dimensional electrophoresis and Emerald 300 glycoprotein staining. α-D-glucosyl, α-D-mannosyl and N-acetylglucosamine residues were detected on slough/eschar-derived proteins. Furthermore, it was demonstrated that the treatment of slough/eschar with ES1 significantly reduced uptake of the carbohydrate-specific stain. Subsequently, α-D-glucosidase, α-D-mannosidase and N-acetylglucosaminidase activities were identified in ES1. Specific chromogenic and fluorogenic substrates and gel filtration chromatography showed that these activities result from distinct enzymes. These activities were mirrored in the removal of α-D-glucosyl, α-D-mannosyl and N-acetylglucosamine residues from proteins of slough/eschar from maggot-treated wounds. These data suggest that maggot glycosidases remove sugars from slough/eschar proteins. This may contribute to debridement, which is ultimately accomplished by a suite of biochemically distinct enzymes present in ES1.

The role of endogenous and exogenous enzymes in chronic wounds: a focus on the implications of aberrant levels of both host and bacterial proteases in wound healing.

Cutaneous wound healing is orchestrated by a number of physiological pathways that ultimately lead to reformation of skin integrity and the production of functional scar tissue. The remodeling of a wound is significantly affected by matrix metalloproteinases (MMPs), which act to control the degradation of the extracellular matrix (ECM). Regulation of MMPs is imperative for wound healing as excessive levels of MMPs can lead to disproportionate destruction of the wound ECM compared to ECM deposition. In addition to human MMPs, bacterial proteases have been found to be influential in tissue breakdown and, as such, have a role to play in the healing of infected wounds. For example, the zinc-metalloproteinase, elastase, produced by Pseudomonas aeruginosa, induces degradation of fibroblast proteins and proteoglycans in chronic wounds and has also been shown to degrade host immune cell mediators. Microbial extracellular enzymes have also been shown to degrade human wound fluid and inhibit fibroblast cell growth. It is now being acknowledged that host and bacterial MMPs may act synergistically to cause tissue breakdown within the wound bed. Several studies have suggested that bacterial-derived secreted proteases may act to up-regulate the levels of MMPs produced by the host cells. Together, these findings indicate that bacterial phenotype in terms of protease producing potential of bacteria should be taken into consideration during diagnostic and clinical intervention of infected wound management. Furthermore, both host MMPs and those derived from infecting bacteria need to be targeted in order to increase the healing capacity of the injured tissue. The aim of this review is to investigate the evidence suggestive of a relationship between unregulated levels of both host and bacterial proteases and delayed wound healing.

Interplay of iron metallobiology, metalloproteinases, and FXIII, and role of their gene variants in venous leg ulcer.

The iron metallobiology has long been suspected as a causal agent in venous leg ulcer (VLU) pathophysiology. However, it was demonstrated only recently that visible iron deposits cause lesions in only some individuals due to functional iron and related gene variants. In this article, the mechanism by which dysregulated iron cycle leads to local iron overload that could generate free radicals or activate a proteolytic hyperactivity on the part of matrix metalloproteinases (MMPs) or else downregulate tissue inhibitors of MMPs is reviewed. Also reviewed is the interplay of other vital factors such as coagulation factor XIII (FXIII), which influences tissue remodeling and angiogenesis, leading to impaired healing of the lesion, whether there exists altered interaction with MMPs or in presence of particular unfavorable single nucleotide polymorphisms.

Mitogen-activated protein kinase p38 pathway in venous ulcer fibroblasts.

OBJECTIVE: Venous ulcer fibroblasts (w-fb) have attenuated growth compared to normal fibroblasts (n-fb). The MAPKp38 pathway mediates stress-responses in various diseases. We hypothesize that p38 pathway is altered in w-fb. METHODS: W-fb were isolated from venous ulcers and n-fb from the ipsilateral thigh. Fibroblasts were analyzed for phosphorylated p38 using immunoblot. The relation between p38 and w-fb proliferation was assessed with SB203580 (p38 inhibitor). Fibroblasts were treated with bFGF, TNF-a, and IL-1 and p38 expression analyzed. RESULTS: Phosphorylated p38 expression was increased in w-fb (AU%=233.5+/-59.7, P=0.039) compared to n-fb (AU%=99.9+/-14.6). W-fb treated with SB203580 demonstrated increased growth compared to untreated w-fb. W-fb treated with bFGF demonstrated decreased p38. TNF-alpha and IL-1beta significantly increase p38 expression. CONCLUSIONS: MAPK p38 is up-regulated in w-fb. Regulation of w-fb proliferation is influenced by p38. Altering the p38 pathway in vivo with growth factors or cytokine inhibition may improve fibroblast proliferation and venous ulcer healing.

Effect of oxidised regenerated cellulose/collagen matrix on proteases in wound exudate of patients with chronic venous ulceration.

Oxidised regenerated cellulose/collagen matrix (ORC/collagen matrix) modifies wound microenvironments by binding and inactivating excess levels of proteases such as elastase, plasmin and gelatinases in wound exudates. To compare levels of the gelatinases matrix metalloproteinase 2 (MMP-2), elastase and plasmin in wound exudates collected from chronic venous insufficiency patients with venous leg ulcers treated with either an ORC/collagen matrix or a standard control therapy. During a 12-week treatment period, wound exudate samples were obtained from a control group of 10 patients treated with a hydrocolloid dressing and a treatment group of 17 patients treated with a combination of ORC/collagen matrix and hydrocolloid dressing. On admission and days 5, 14 and every subsequent 14th day, ulcers were photographed to determine healing rate and changes in ulcer appearance, and MMP-2 concentration and the gelatinase, elastase and plasmin activities were analysed from wound exudates. The patients treated with ORC/collagen matrix showed a significant decrease in elastase, plasmin and gelastinase activity as compared with the control group, with no significant difference in the MMP-2 concentrations between the two groups. The results show a significant and immediate reduction in protease activity in wound exudates from venous leg ulcers treated with ORC/collagen.

Effect of collagen turnover and matrix metalloproteinase activity on healing of venous leg ulcers.

BACKGROUND: The presence of fibrous tissue in poorly healing venous leg ulcers suggests abnormal collagen metabolism. The aim was to determine whether there were differences in collagen turnover and matrix metalloproteinase (MMP) activity between ulcers that healed, those that did not heal and normal skin. METHODS: Biopsies were taken from the ulcers of 12 patients whose venous ulcers went on to heal and 15 patients whose ulcers failed to heal despite 12 months of compression bandaging. Biopsies were taken from 15 normal controls. Collagen turnover (collagen III N-terminal propeptide (PIIINP) and degraded collagen), and total MMP, MMP-1 and MMP-3 activities were measured. RESULTS: PIIINP and degraded collagen levels were higher in ulcers that healed compared with lesions that failed to heal (P = 0.005 and P < 0.001 respectively) and normal skin (P = 0.003 and P < 0.001). MMP-1 activity was also higher in healing ulcers than resistant ulcers (P < 0.001) and normal skin (P < 0.001). Significantly more total MMP activity was present in all ulcers than in normal skin (P < 0.001), but there was no difference in total MMP (and MMP-3 activity) between ulcers that healed and those that did not. CONCLUSION: Rapidly healing venous leg ulcers had increased collagen turnover and MMP-1 activity, which appeared to differentiate them from those that failed to heal within 12 months.

Multiplexed analysis of matrix metalloproteinases in leg ulcer tissue of patients with chronic venous insufficiency before and after compression therapy.

Elevated matrix metalloproteinases (MMP) levels have been implicated in the pathogenesis of chronic venous insufficiency ulcers. Quantitative measurements of a broad range of MMP proteins in human tissue treated with compression bandaging have not been reported. The goal of this study was to determine the expression of a wide range of proteases in untreated venous leg ulcer tissue and the changes in these levels after 4 weeks of high-strength compression therapy. Twenty-nine limbs with new or untreated chronic venous insufficiency and leg ulceration received therapy for 4 weeks with sustained high compression bandaging. Biopsies were obtained from healthy tissue and from ulcerated tissue before and after therapy. A novel multiplexed protein assay was used to measure multiple MMPs in a single sample. MMP protein activity, TIMP protein levels, and gene expression levels were also addressed. MMP1, 2, 3, 8, 9, 12, and 13 protein levels were elevated in ulcer tissue compared with healthy tissue. MMP8 and 9 were highly expressed in ulcer tissue. MMP3, 8, and 9 significantly decreased following treatment. Reduction in the levels of MMP1, 2, and 3 was associated with significantly higher rates of ulcer healing at 4 weeks. We conclude that compression therapy results in a reduction of the pro-inflammatory environment characterizing chronic venous ulcers, and ulcer healing is associated with resolution of specific elevated levels of protease expression.

Matrix metalloproteinases in peripheral vascular disease.

Matrix metalloproteinases (MMPs) are extracellular matrix-modifying enzymes that are important in many physiologic and pathologic vascular processes. Dysregulation of MMP activity has been associated with common vascular diseases such as atherosclerotic plaque formation, abdominal aortic aneurysms, and critical limb ischemia. For this reason, MMPs have become an important focus for basic science studies and clinical investigations by vascular biology researchers. This article reviews the recent literature, summarizing our current understanding of the role of MMPs in the pathogenesis of various peripheral vascular disease states. In addition, the importance of MMPs in the future diagnosis and treatment of peripheral vascular disease is discussed.

Mitogen-activated protein kinase pathway regulates cell proliferation in venous ulcer fibroblasts.

Venous ulcer fibroblasts have been demonstrated to have low growth rates in response to platelet-derived growth factor (PDGF). Mitogen-activated protein kinase (MAPK) is an important signal transduction mechanism that regulates growth, differentiation, and apoptosis in eukaryotic cells. PDGF binds PDGF receptors that activate a multitiered signaling cascade involving MAPK. We hypothesize that the growth regulation in venous ulcer fibroblasts is dependent on the MAPK extracellular signal-regulated kinase (ERK) pathway in the presence of PDGF. Fibroblasts (fb) were isolated from 8 patients with venous ulcers (w-fb) and the normal skin (n-fb) of the ipsilateral thigh via punch biopsies. Fb were plated at 1,500 cells/dish and treated with PDGF-AB (10 ng/mL) for 15 days. Growth rates were determined. Immunoblot analysis of MAPK ERK for n-fb and w-fb were analyzed. To determine if PDGF-stimulated w-fb and n-fb utilized the MAPK ERK pathway in a dependent manner, the upstream kinase MAPK kinase 1 (MEK 1) was inhibited by PD 98059. In addition, fb were treated with chronic venous ulcer wound fluid (WF) to study its effect on MAPK ERK. In the presence of PDGF, growth rates were substantially lower in w-fb than in n-fb, and MAPK was activated in 6/8 w-fb and in only 2/8 n-fb. Fibroblasts expressing MAPK had significantly reduced cell proliferation compared to fibroblasts not expressing MAPK (p = 0.023). PD 98059 significantly inhibited w-fb and n-fb cell proliferation from basal level, which was reversible with addition of PDGF. In neonatal fibroblasts WF demonstrated inhibition of MAPK ERK over time and addition of PD98059 was not additive. This study suggests that the MAPK ERK pathway is important for cell proliferation in venous ulcer fibroblasts. In the presence of PDGF, fibroblasts with decreased growth rate express MAPK, and proliferation is further abrogated with addition of MEK 1 inhibitor, suggesting the importance of the MAPK ERK pathway regulating w-fb and n-fb proliferation. Although the majority of w-fb activated the MAPK ERK pathway in the presence of PDGF, proliferation was significantly attenuated, indicating that other MAPK inhibitory pathways are competing. Venous ulcer wound fluid directly inhibits the MAPK ERK pathway, suggesting that the venous ulcer wound environment has negative trophic factors that effect fibroblasts proliferation and ulcer healing.

The impact of differential expression of extracellular matrix metalloproteinase inducer, matrix metalloproteinase-2, tissue inhibitor of matrix metalloproteinase-2 and PDGF-AA on the chronicity of venous leg ulcers.

INTRODUCTION: Alteration in the expression of extracellular matrix metalloproteinase inducer (EMMPRIN), matrix metalloproteinase-2 (MMP-2), tissue inhibitors of matrix metalloproteinases (TIMP-2) and platelet derived growth factor (PDGF-AA) may contribute to poor healing in venous leg ulcers. AIM: The aim of this study is to determine the expression of EMMPRIN, MMP-2, TIMP-2 and PDGF-AA in the ulcer exudates and perivascular tissue of healing and non-healing chronic venous ulcers. PATIENTS, MATERIALS AND METHODS: Forty patients with chronic venous ulcers were included in this study, with a mean age of 60 years. Eleven patients were males and 29 were females. All patients had normal ankle brachial index and a venous ulcer of at least 8 weeks duration. Immuno-histochemistry using monoclonal antibodies to PDGF-AA, MMP-2, TIMP-2 and EMMPRIN was carried out on paraffin embedded punch biopsy skin specimens from the ulcer edge. Enzyme linked immunosorbent assay for PDGF, MMP-2 and TIMP-2 were carried out on wound fluids collected from patients. The ulcer size and character at the initial assessment and after 8 weeks were assessed to determine the status of ulcer healing. RESULTS: No significant difference was seen in the expression of TIMP-2, MMP-2 and EMMPRIN between the two groups. However, in the non-healing group high levels of MMP-2 and low levels of TIMP-2 in the wound fluid suggest a strong correlation of these two markers in the state of healing. Analysis of wound fluid by ELISA demonstrated high PDGF-AA in the healing group (p = 0.021). Significantly increased levels of PDGF-AA (p<0001) was noted in the perivascular area on immuno-histochemistry of healing ulcers. These data suggest that PDGF-AA plays an important role in healing of venous ulcers. CONCLUSION: Non-healing venous ulcers are associated with greater activity MMP-2 activity. The ratio of MMPs to their inhibitors TIMPs, dictate the rate of healing of the ulcers. PDGF-AA activity is associated with ulcer healing, though the mechanism is unclear. EMMPRIN expression in chronic venous ulcers probably parallels the chronicity of the condition rather than propagate it. However, further studies with larger samples are needed.

Mast cell tryptase and chymase in chronic leg ulcers: chymase is potentially destructive to epithelium and is controlled by proteinase inhibitors.

BACKGROUND: Numerous mast cells are present in chronic leg ulcers. Tryptase and chymase are the major mediators of mast cells, but their significance is mostly dependent on their activity. In addition, the proteinases may affect ulcer epithelialization. OBJECTIVES: To study levels and activity of tryptase and chymase in wash samples and biopsies from chronic leg ulcers and the possible effect of these proteinases on keratinocyte growth and adherence. METHODS: Wash samples were taken from 16 patients and a superficial shave biopsy was taken in eight of these patients; a second biopsy series was obtained from the edge of chronic venous leg ulcers (n = 6). RESULTS: Significant levels of soluble tryptase activity and histamine, but low levels of chymase activity, were measured in wash samples from chronic ulcers. No tryptase-inhibiting activity, but clear chymase-inhibiting activity, was detected in the wash samples. In superficial wound bed biopsies, relatively marked levels of chymase activity together with histamine and tryptase activity were detected. In the second biopsy series, about 80% of the mast cells belonged to the MC(TC) type (tryptase- and chymase-immunopositive). However, about 55-61% of the chymase-immunopositive cells displayed chymase activity and 64 +/- 17% of the tryptase-positive cells revealed immunoreactivity of alpha(1)-antichymotrypsin. As the activity of chymase and tryptase was detected in the ulcer base in a ratio of 1:8, a preparation containing both chymase and tryptase was partially purified from human skin yielding a similar activity ratio of 1:11-13. Treatment of fibronectin-coated plastic surfaces with this preparation decreased the adherence of cultured human keratinocytes, this effect being attributable mainly to chymase. In 2-day cultures using growth factor/serum-deficient low- or high-calcium medium, the tryptase-chymase preparation inhibited the slow growth and at higher concentrations it even induced detachment of keratinocytes. This effect was attributed to chymase, and it was partially regulated by heparin and histamine. CONCLUSIONS: Even though chymase is partially inactivated in chronic leg ulcers, accumulated mast cells in the close proximity of the epithelium edge and their chymase may impair keratinocyte adherence and migration.

[The role of matrix metalloproteinases in venous leg ulcers development]. / Rola metaloproteaz macierzy w powstawaniu owrzodzen zylnych goleni.

Venous leg ulcers are one of the most serious therapeutic and economic problems of current medicine. In spite of the rapid progress in the diagnostics of venous disorders, the pathogenesis of venous leg ulcers remains unclear. Current medicine describes many theories that try to explain the patophysiology of venous ulceration. Recent studies have revealed the role of extracellular matrix degradation processes in venous leg ulcers. The family of enzymes classified as matrix metalloproteinases (MMPs) play the leading role in these processes. The extensive knowledge of MMPs and the role played by them in venous leg ulcers and wound healing may result in development of the new methods of management with many vascular disorders.

Elevated expression of extracellular matrix metalloproteinase inducer (CD147) and membrane-type matrix metalloproteinases in venous leg ulcers.

BACKGROUND: Matrix metalloproteinases (MMPs) contribute to matrix remodelling in venous leg ulcers. Extracellular MMP inducer (EMMPRIN; CD147) has been reported to increase MMP expression, and membrane type 1 MMP (MT1-MMP) has been implicated in the activation of MMPs. OBJECTIVES: To examine whether and to what degree EMMPRIN, MMP-2, MT1-MMP and membrane type 2 MMP (MT2-MMP) are expressed in venous leg ulcers as well as the association with MMP activity. METHODS: EMMPRIN, MMP-2, MT1-MMP and MT2-MMP were analysed by zymography and immunohistochemistry in biopsies from healthy skin and lesional tissue from venous leg ulcers. RESULTS: Zymography provided direct evidence of increased proteolytic activity of MMP-2 in lesional skin in comparison with healthy controls. Immunostaining showed intense expression of EMMPRIN, MMP-2, MT1-MMP and MT2-MMP in dermal structures of venous leg ulcers, whereas only EMMPRIN and MMP-2 showed elevated expression in perivascular regions. Our findings indicate that venous leg ulcers are characterized by elevated expression of EMMPRIN, MMP-2, MT1-MMP and MT2-MMP. The immunohistological findings of skin alterations reflect the dynamic process of activation of soluble and membrane-bound MMPs, which may be highly induced by EMMPRIN. CONCLUSIONS: These data suggest for the first time that membrane-bound MMPs may favour enhanced turnover of the extracellular matrix and support unrestrained MMP activity in venous leg ulcers.