International Wound Journal ISSN 1742-4801
ORIGINAL ARTICLE
Extracellular matrix assessment of infected chronic venous leg ulcers: role of metalloproteinases and inflammatory cytokines Raffaele Serra1,2 , Raffaele Grande1 , Gianluca Buffone1 , Vincenzo Molinari1 , Paolo Perri1 , Aldina Perri1 , Bruno Amato3 , Manuela Colosimo4 & Stefano de Franciscis1,2 1 Interuniversity Center of Phlebolymphology, International Research and Educational Program in Clinical and Experimental Biotechnology, University Magna Graecia of Catanzaro, Catanzaro, Italy 2 Department of Medical and Surgical Science, University Magna Gracia of Catanzaro, Catanzaro, Italy 3 Department of Clinical Medicine and Surgery, University of Naples Federico II, Napoli, Italy 4 Department of Microbiology, University Hospital of Milan, Milan, Italy Key words Chronic venous leg ulcers; Cytokines; Infection; Metalloproteinases; Wound healing
Serra R, Grande R, Buffone G, Molinari V, Perri P, Perri A, Amato B, Colosimo M, de Franciscis S. Extracellular matrix assessment of infected chronic venous leg ulcers: role of metalloproteinases and inflammatory cytokines. Int Wound J 2014; doi: 10.1111/iwj.12225
Correspondence to
Abstract
Prof. R Serra, MD, PhD Department of Medical and Surgical Science School of Medicine University Magna Graecia of Catanzaro Viale Europa, Localita` Germaneto 88100 Catanzaro Italy E-mail: [email protected]
Chronic venous ulcer (CVU) represents a dreaded complication of chronic venous disease (CVD). The onset of infection may further delay the already precarious healing process in such lesions. Some evidences have shown that matrix metalloproteinases (MMPs) are involved and play a central role in both CVUs and infectious diseases. Two groups of patients were enrolled to evaluate the expression of MMPs in infected ulcers and the levels of inflammatory cytokines as well as their prevalence. Group I comprised 63 patients (36 females and 27 males with a median age of 68·7 years) with infected CVUs, and group II (control group) comprised 66 patients (38 females and 28 males with a median age of 61·2 years) with non-infected venous ulcers. MMP evaluation and dosage of inflammatory cytokines in plasma and wound fluid was performed by means of enzyme-linked immunosorbent assay test; protein extraction and immunoblot analysis were performed on biopsied wounds. The first three most common agents involved in CVUs were Staphylococcus aureus (38·09%), Corynebacterium striatum (19·05%) and Pseudomonas aeruginosa (12·7%). In this study, we documented overall higher levels of MMP-1 and MMP-8 in patients with infected ulcers compared to those with uninfected ulcers that showed higher levels of MMP-2 and MMP-9. We also documented higher levels of interleukin (IL)-1, IL-6, IL8, vascular endothelial growth factor and tumour necrosis factor-alpha in patients with infected ulcers with respect to those with uninfected ulcers, documenting a possible association between infection, MMP activation, cytokine secretions and symptoms. The present results could represent the basis for further studies on drug use that mimic the action of tissue inhibitors of metalloproteinases in order to make infected CVU more manageable.
Introduction
Chronic venous disease (CVD), widespread in western countries (up to 57% in males and 77% in females), plays a major role in quality of life and work activities of affected patients (1–6). CVD is related to several complications that may be classified through Clinical-EtiologyAnatomy-Pathophysiology (CEAP) classification (7). Chronic venous ulcers (CVUs) are the most common type of wounds affecting the lower limbs (8–12) and they represent a common complication of CVD. CVUs represent the pathophysiological expression of an inflammatory process orchestrated by specific
and non-specific immune response; inflammatory cells provide growth factors and stimulate the deposition of matrix proteins
Key Messages • chronic venous ulceration (CVU) can result in several
complications, including the onset of infection, causing increasing pain and persistent ulceration • matrix metalloproteinases (MMPs) and inflammatory cytokines play an important role in both
© 2014 The Authors International Wound Journal © 2014 Medicalhelplines.com Inc and John Wiley & Sons Ltd doi: 10.1111/iwj.12225
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ECM, CVU, MMPs and cytokines Table 1 Characteristics of patients
pathophysiological mechanism of CVUs and infectious diseases; when bacteria begin replication and increase their metabolic activity, the resulting by-products, such as endotoxins and MMPs, negatively impact all phases of wound healing – moreover, inflammatory cytokines are important mediators of protein catabolism in infectious diseases • in our study, we documented higher levels of MMP-1 and MMP-8 and the cytokines, IL-1, IL-6, IL-8, VEGF and TNF-α, in patients with infected ulcers compared to those with uninfected ulcers • the present results could represent the basis for further studies on drugs that could lower the levels of MMPs and make infected CVUs more manageable
and phagocytose debris. CVUs are chronic skin lesions and may be complicated by infections (13,14); the risk of wound infection is increased when an imbalance occurs between local factors favouring bacterial growth and integrity of the immune defences. These lesions may become non-healing chronic wounds as long as the antigens from microorganisms remain. Several studies have shown that the inflammatory process characterising CVUs is associated with the presence of matrix metalloproteinase (MMP) activation (15–18). MMPs are related with extracellular matrix (ECM), a network of interlacing macromolecules that forms a supporting structure for vascular wall and skin integrity. ECM is dynamically maintained by the action of MMPs (which degrade ECM proteins) and their inhibitors (tissue inhibitors of MMPs, TIMPs) (19). Evidences have shown that MMPs play a central role in infectious diseases (20–23) also through ECM degradation, which favours the migration of immune cells from the bloodstream to the site of inflammation (24–26). The chronic exposure of CVUs to the environment, associated with the individual susceptibility of patients, predisposes the onset of infectious processes that worsen the prognosis for cure (27). In this study, we evaluated the expression of MMPs in infected ulcers and the levels of inflammatory cytokines. Materials and methods Study design
This study was approved by the Institutional Review Board–Independent Ethics Committee (IRB-IEC) of Interuniversity Center of Phlebolymphology – International Research and Educational Program in Clinical and Experimental Biotechnology – at University Magna Graecia of Catanzaro. Prior to the study, all participants were informed about the aim, procedures, risks and benefits of the study and a written informed consent was obtained. We conducted an open-label, parallel-group study between January 2011 and December 2012 in two Clinical Departments of Catanzaro and Naples. CVUs are included in stage 6 of CEAP classification (7). At the time of admission, patients’ medical history was recorded, and clinical examination, ulcer duplex ultrasonography and computed haemodynamic 2
Number of enrolled patients Male/female Age (years) Range Duplex ultrasound findings Superficial vein incompetence Perforating vein incompetence Superficial + perforating vein incompetence Ulcer features Ulcer area (range) Ulcer area (mean) Leucocytes: median/mm3 Concomitant conditions Diabetes Obesity Arterial hypertension COPD Malignancy Length of hospital stay (days ± SD)
Group I: patients with infected CVUs
Group II: patients with uninfected CVUs
63 27/36 59·4 ± 9 47–73
66 28/38 54·0 ± 8·6 45–70
11 (17·46%)
25 (37·88%)
25 (38·68%)
20 (30·30%)
27 (42·86%)
21 (31·81%)
2·5–19·9 cm2 12·7 cm2 14 600
2·1–17·8 cm2 10·5 cm2 11 000
14 10 21 6 12 9·4 ± 7·8
16 13 18 9 10 3·2 ± 9
CVU, chronic venous ulcer; COPD, chronic obstructive pulmonary disease.
mapping (28,29) were performed. Moreover, a blood sample was drawn at the time of admission for laboratory evaluation. The inclusion criteria were presence of infected venous ulcers, ankle-brachial pressure index (ABPI) >0·9 and, when associated with blood hypertension, a diastolic arterial pressure below 95 mmHg. The exclusion criteria were presence of arterial disease, connective tissue disorders including rheumatoid arthritis, blood disorders or cancer and ischaemia (ABPI < 0·9), smoking, systemic diseases, diabetes mellitus, use of any medications within the previous 7 days that could impair wound healing, and topical or systemic antimicrobial therapy. Patient characteristics are shown in Table 1. MMP evaluation in plasma and wound fluid
MMPs were measured in fluids of healing wounds and non-healing ulcers by enzyme-linked immunosorbent assay (ELISA), in agreement with previous studies (4,15–18,30–34). Protein extraction and immunoblot analysis Western blot was performed to estimate the expression of MMP-1, MMP-2, MMP-8 and MMP-9 in tissues, in agreement with previous studies (4,15–18,30,32–34). Wounds were subsequently biopsied (T = 1) under 1% lidocaine local anaesthesia and with full sterile precautions. The biopsy was made at a point equidistant from the centre and edge of the ulcer. Our experience with biopsies in this
© 2014 The Authors International Wound Journal © 2014 Medicalhelplines.com Inc and John Wiley & Sons Ltd
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patient population indicates that it is well tolerated by the subject and does not influence healing outcomes in venous ulcers. One biopsy was immediately placed into a sterile collection container and sent for quantitative (microbiology) culture. In both group I and group II, the biopsies obtained at the time of wound bed preparation (T = 1) were used for Western blot analysis in 2 ml of tissue protein extraction reagent (25 mM Bicine and 150 mM sodium chloride, pH = 7·6; Thermo Scientific, Cambridge, UK). Protein concentrations were determined and lysates were stored at 80◦ C. For Western blot, protein extracts were separated on a 12·5% sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes as previously described. Immunoblotting was performed using anti-MMP-1, anti-MMP-2, anti-MMP-8 and anti-MMP-9 monoclonal antibodies and expressed as arbitrary units. All experiments were performed in triplicate.
Biopsy
Biopsies were obtained under local anaesthesia with 2% lidocaine from the border area of the ulcer and from similar anatomical site of non-infected subjects. In order to eliminate bacterial infection, swabs for microbiology studies were obtained from all cases at the same time when biopsy was taken. The obtained tissue measured 10 mm length × 5 mm width, comprising the ulcer edge and surrounding skin. Each biopsy was divided into two halves: one was fixed in formaldehyde buffer with 10% glycine, while the other half was kept in RNAlater (Qiagen, Duesseldorf, Germany). The biopsies were embedded in paraffin, cut into 4-mm sections and stained with picrosirius. Collagen fibres were quantified under a microscope at 160× magnification using a millimetre ruler on the eyepiece in a 10-mm field. The thickness of each collagen fibre present in this field was measured. The quantity of collagen fibres was determined as the sum of the fibre thicknesses divided by 10 mm. For primary cell culture, the tissue was kept in Dulbecco’s modified Eagle’s medium and HAM F-12 for no longer than 2 hours. Biopsies were fixed for 24 hours and embedded in paraffin.
Immunohistochemical analysis
Sections of 4-mm thick were mounted on silane-coated slides, deparaffinised and the endogenous peroxidase was quenched with 0·03% H2 O2 in absolute methanol. Then, the sections were washed and blocked with phosphate-buffered saline supplemented with 2% goat serum. Skin sections were incubated with one of the following antibodies for 18 hours, anti-LL-37, anti-HBD-1, anti-HBD-2, anti-HBD-3, anti-HBD4 or anti-HNP-1, and then washed and incubated for 2 hours with anti-goat or anti-rabbit IgG biotin-labelled antibody. Bound antibodies were detected with avidin–biotin peroxidase and counterstained with haematoxylin. The whole biopsy area was taken for quantification. At 100× magnification, all negative or positive cells from the epidermis were counted using an image analyser.
ECM, CVU, MMPs and cytokines
The slides were counterstained with Harris haematoxylin. For all markers, the sections were analysed under a microscope at 400× magnification and the quantification was performed as the mean of the sum of labelled cells in each analysed field divided by the number of fields. The same procedure was used in the dermis. Cytokines analysis
An ELISA Kit (Human ELISA System, Amersham Pharmacia Biotech, Buckinghamshire, UK) was used to determine the concentration of interleukin (IL)-1, IL-6, IL-8, vascular endothelial growth factor (VEGF) and tumour necrosis factoralpha (TNF-α) in both plasma and wound fluid, in agreement with previous studies (15,18) Statistical analysis
Data analysis was performed using SigmaStats 3.5 (STACON, Witzenhausen, Germany). Kruskal–Wallis analysis of variance (ANOVA) was performed for all statistical comparisons between groups, and the Mann–Whitney U -test was performed for individual comparisons. All data are expressed as mean ± standard error of the mean (SEM) and P < 0·05 was considered significant. Results Patients
Enrolled patients were randomised into two groups. Group I comprised 63 patients (36 females and 27 males with a median age of 68·7 years) with infected CVUs, evidence of venous reflux at duplex scanning, and group II (control group) comprised 66 patients (38 females and 28 males with a median age of 61·2 years) with non-infected venous ulcers. Microbiological agents involved in CVUs
The most common agents involved in CVUs were Staphylococcus aureus (38·09%), Corynebacterium striatum (19·05%) and Pseudomonas aeruginosa (12·7%); remaining bacterial strains were as follows: Escherichia coli (3·17%), Staphylococcus epidermidis (4·76%), Alcaligenes faecalis (1·59%), Stenotrophomonas maltophilia (3·17%), Enterococcus faecium (4·76%), Streptococcus pyogenes (6·35%), Acinebacterium haemoliticus (3·17%), Providencia rettgeri (1·59%) and Staphylococcus simulans (1·59%) (Table 2). MMP values in plasma and tissues
ELISA test and Western blot analysis documented higher levels of MMP-1 and MMP-8 in group I, and MMP-2 and MMP-9 in group II (Figures 1 and 2). Dosage of inflammatory cytokines
ELISA test revealed higher levels of IL-1, IL-6, IL-8, VEGF and TNF-α in plasma and wound fluid of patients with infected CVU (group I) compared to those with uninfected CVU (group II) (Figure 3).
© 2014 The Authors International Wound Journal © 2014 Medicalhelplines.com Inc and John Wiley & Sons Ltd
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ECM, CVU, MMPs and cytokines
Staphylococcus aureus Corinebacterium striatum Pseudomonas aeruginosa Escherichia coli Staphylococcus epidermidis Alcaligenes faecalis Stenotrophomonas maltophilia Enterococcus faecium Streptococcus pyogenes Acinebacterium haemoliticus Providencia rettgeri Staphylococcus simulans
MMPs plasma levels (ng/ml)
A
**
24 (38·09) 12 (19·05) 8 (12·7) 2 (3·17) 3 (4·76) 1 (1·59) 2 (3·17) 3 (4·76) 4 (6·35) 2 (3·17) 1 (1·59) 1 (1·59)
group I
group II
**
30 25 20
group I
group II
**
100 80 60 40 20 0
B
1200 1000
MMP-1 **
MMP-8 group I
group II
800 600 **
400 200 0
MMP-2
10
0
1200
MMP-1 **
MMP-8 group I
MMP-9
Figure 2 Tissue levels of matrix metalloproteinases (MMP)-1 and MMP8 (A) and MMP-2 and MMP-9 (B) measured through Western blot in enrolled patients at the time of admission. Group I: patients with infected ulcers; group II: patients with uninfected ulcers. **P < 0·01.
group II
1200
**
group I
group II
1000
1000 800
Plasma levels (pg/mL)
MMPs plasma levels (ng/ml)
**
120
15
5
B
MMPs tissues expression (Arbitrary Units)
N (%)
Species name
A
MMPs plasma levels (ng/mL)
Table 2 Percentage distribution of bacterial species in the samples of enrolled patients
600 **
400 200
**
**
**
800 600 400 200
0
MMP-2
MMP-9
0
Figure 1 Plasma levels of matrix metalloproteinases (MMP)-1 and MMP-8 (A) and MMP-2 and MMP-9 (B) measured through enzymelinked immunosorbent assay (ELISA) in enrolled patients at the time of admission. Group I: patients with infected ulcers; group II: patients with uninfected ulcers. **P < 0·01. Kruskal–Wallis analysis of variance (ANOVA) test was performed to compare plasma MMP-8 values between two groups.
Discussion
In this study, we evaluated the expression of MMPs and cytokines in patients with infected and non-infected CVUs. Wound healing progresses through several phases: (i) haemostasis; (ii) inflammation (removal of debris, control of infection and clearance of inflammation); (iii) proliferation 4
VEGF
TNF-alpha
IL-6
IL-8
Figure 3 Cytokine plasma levels of vascular endothelial growth factor (VEGF), tumour necrosis factor-alpha (TNF-α), interleukin (IL)-1, IL-6 and IL-8 measured through enzyme-linked immunosorbent assay (ELISA) in enrolled patients at the time of admission. Group I: patients with infected ulcers; group II: patients with uninfected ulcers. **P < 0·01.
(angiogenesis, deposition of granulation tissue and contraction); and (iv) remodelling (remodelling of the connective tissue matrix, and maturation) (35). The normal wound healing process is a complex physiological event that depends on interactions between epidermal keratinocytes, dermal fibroblasts, Langerhans cells, endothelial cells and fibroblasts, and is coordinated via complex cell–cell and cell–matrix
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interactions (36). CVUs are lesions characterised by prolonged inflammation, a defective wound matrix and failure of reepithelialisation; these factors facilitate the colonisation by major pathogens, particularly Gram-positive and Gram-negative bacteria. This study showed a high prevalence of S. aureus infections, followed by P. aeruginosa and C. striatum. These bacterial species had evolved numerous mechanisms to evade host defence strategies employed by neutrophils, including the ability to modulate normal neutrophil turnover, a process critical to the resolution of acute inflammation. Gender, age (37), defence mechanisms (33), CVU severity [according to CEAP classification (7)] and hormonal and metabolic status of the host are among the factors influencing the vital activity of commensal. Wound infection advances in stages from contaminated, colonised, critically colonised to infected wounds. When bacteria begin replication and increase their metabolic activity, the resulting by-products, such as endotoxins and MMPs, negatively impact all phases of wound healing. MMPs play a central role in infectious diseases when the host immune system is challenged by an invading organism, facilitating the recruitment of leucocytes from the bloodstream; these migrate to the site of infection for eradication of the pathogen (by proteolysis of the basement membrane) and for modulating the inflammatory response (31). In this study, we documented higher levels of MMP-1 and MMP-8 in patients with infected ulcers compared to those with uninfected ulcers that showed higher levels of MMP-2 and MMP-9. These results document a different role of MMPs during the infection, and these results may be in agreement with other studies (34,38,39) that documented that MMP-1 is increased upon stimulation with inflammatory cytokines such as IL-1β, TNF-α and IL-6 showing a correlation between MMPs and cytokines. Inflammatory cytokines, such as TNF-α, IL-1 and IL-6, are important mediators of protein catabolism in infectious diseases (40–43) such as infected CVUs. Moreover, polymorphonuclear granulocytes produce MMP-8, a neutrophil collagenase, which is pivotal for initiation and consequent resolution of inflammation, presumably by altering chemotactic gradients (44). TNF-α and IL-1 play a central role in the regulation of collagen metabolism in fibroblasts; addition of recombinant TNF to cultured human fibroblasts significantly decreased both collagen gene transcription and peptide synthesis (45,46). TNF also modulates collagen metabolism by increasing the synthesis of collagenase, an enzyme that digests collagen (47). In this study, we documented higher levels of IL-1, IL-6, IL-8, VEGF and TNF-α in plasma and wound fluid of patients with infected ulcers compared to those with uninfected ulcers, documenting a possible association between infection, MMP activation, cytokine secretions and symptoms. Patients with severe or persistent infections are at particularly high risk for problems related to poor wound healing and these problems require either reoperation or prolonged medical care (48,49). Previously, in an experimental study, we documented that corticosteroid may be able to modulate the expression of TNF-α and inflammatory cytokines (50) in epithelial cells; therefore, the present results could represent the basis for further studies on drug use that mimic the action of TIMP-1,
ECM, CVU, MMPs and cytokines
a tissue inhibitor of MMPs (51); these could lower the levels of MMPs and make CVUs more manageable. Acknowledgements
This study received no funding. The authors declare that they have no competing interests. References 1. Robertson L, Evans C, Fowkes FGE. Epidemiology of chronic venous disease. Phlebology 2008;23:103–11. 2. Beebe-Dimmer JL, Pfeifer JR, Engle JS, Schottenfeld D. The epidemiology of chronic venous insufficiency and varicose veins. Ann Epidemiol 2005;15:175–84. 3. Serra R, Grande R, Buffone G, Costanzo G, Damiano R, de Franciscis S. Chronic venous disease is more aggressive in patients with varicocele. Acta Phlebol 2013;14:57–60. 4. Serra R, Buffone G, Costanzo G, Montemurro R, Scarcello E, Stillitano DM, Damiano R, de Franciscis S. Altered metalloproteinase-9 expression as the least common denominator between varicocele, inguinal hernia and chronic venous disorders. Ann Vasc Surg 2013, In press, pii: S0890-5096(13)00576-1. DOI: 10.1016/j.avsg.2013.07.026. 5. Serra R, Buffone G, de Franciscis A, Mastrangelo D, Molinari V, Montemurro R, de Franciscis S. A genetic study of chronic venous insufficiency. Ann Vasc Surg 2012;26:636–42. 6. Serra R, Buffone G, Costanzo G, Montemurro R, Perri P, Damiano R, de Franciscis S. Varicocele in younger as risk factor for inguinal hernia and for chronic venous disease in older: preliminary results of a prospective cohort study. Ann Vasc Surg 2013;27:329–31. 7. Ekl¨of B, Rutherford RB, Bergan JJ, Carpentier PH, Gloviczki P, Kistner RL, Meissner MH, Moneta GL, Myers K, Padberg FT, Perrin M, Ruckley CV, Smith PC, Wakefield TW, American Venous Forum International Ad Hoc Committee for Revision of the CEAP Classification. Revision of the CEAP classification for chronic venous disorders: consensus statement. J Vasc Surg 2004;40:1248–52. 8. Serra R, Buffone G, Molinari V, Montemurro R, Perri P, Stillitano DM, Amato B, de Franciscis S. Low molecular weight heparin improves healing of chronic venous ulcers especially in the elderly. Int Wound J 2013, In press. DOI: 10.1111/iwj.12071. 9. de Franciscis S, De Sarro G, Longo P, Buffone G, Molinari V, Stillitano DM, Gallelli L, Serra R. Hyperhomocysteinaemia and chronic venous ulcers. Int Wound J 2013, In press. DOI: 10.1111/iwj.12042. 10. Serra R, Buffone G, de Franciscis A, Mastrangelo D, Vitagliano T, Greco M, de Franciscis S. Skin grafting followed by low-molecularweight heparin long-term therapy in chronic venous leg ulcers. Ann Vasc Surg 2012;26:190–7. DOI: 10.1016/j.avsg.2011.04.008. 11. de Franciscis S, Grande R, Buffone G, Serra R. Chronic venous ulceration of the lower limbs and thrombosis. Acta Phlebol 2013, In press. 12. Gasbarro V, Amato B, Izzo M, Manfrini A, Buffone G, Grande R, Serra R, de Franciscis S. Quercetin and chronic venous ulceration of the lower limbs. Acta Phlebol 2013;14:61–5. 13. Edwards R, Harding KG. Bacteria and wound healing. Curr Opin Infect Dis 2004;17:91–6. 14. Debats IB, Booi D, Deutz NE, Buurman WA, Boeckx WD, van der Hulst RR. Infected chronic wounds show different local and systemic arginine conversion compared with acute wounds. J Surg Res 2006;134:205–14. 15. Serra R, Buffone G, Falcone D, Molinari V, Scaramuzzino M, Gallelli L, de Franciscis S. Chronic venous leg ulcers are associated with high levels of metalloproteinases-9 and neutrophil gelatinaseassociated lipocalin. Wound Repair Regen 2013;21:395–401. DOI: 10.1111/wrr.12035.
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ORIGINAL ARTICLE
Extracellular matrix assessment of infected chronic venous leg ulcers: role of metalloproteinases and inflammatory cytokines Raffaele Serra1,2 , Raffaele Grande1 , Gianluca Buffone1 , Vincenzo Molinari1 , Paolo Perri1 , Aldina Perri1 , Bruno Amato3 , Manuela Colosimo4 & Stefano de Franciscis1,2 1 Interuniversity Center of Phlebolymphology, International Research and Educational Program in Clinical and Experimental Biotechnology, University Magna Graecia of Catanzaro, Catanzaro, Italy 2 Department of Medical and Surgical Science, University Magna Gracia of Catanzaro, Catanzaro, Italy 3 Department of Clinical Medicine and Surgery, University of Naples Federico II, Napoli, Italy 4 Department of Microbiology, University Hospital of Milan, Milan, Italy Key words Chronic venous leg ulcers; Cytokines; Infection; Metalloproteinases; Wound healing
Serra R, Grande R, Buffone G, Molinari V, Perri P, Perri A, Amato B, Colosimo M, de Franciscis S. Extracellular matrix assessment of infected chronic venous leg ulcers: role of metalloproteinases and inflammatory cytokines. Int Wound J 2014; doi: 10.1111/iwj.12225
Correspondence to
Abstract
Prof. R Serra, MD, PhD Department of Medical and Surgical Science School of Medicine University Magna Graecia of Catanzaro Viale Europa, Localita` Germaneto 88100 Catanzaro Italy E-mail: [email protected]
Chronic venous ulcer (CVU) represents a dreaded complication of chronic venous disease (CVD). The onset of infection may further delay the already precarious healing process in such lesions. Some evidences have shown that matrix metalloproteinases (MMPs) are involved and play a central role in both CVUs and infectious diseases. Two groups of patients were enrolled to evaluate the expression of MMPs in infected ulcers and the levels of inflammatory cytokines as well as their prevalence. Group I comprised 63 patients (36 females and 27 males with a median age of 68·7 years) with infected CVUs, and group II (control group) comprised 66 patients (38 females and 28 males with a median age of 61·2 years) with non-infected venous ulcers. MMP evaluation and dosage of inflammatory cytokines in plasma and wound fluid was performed by means of enzyme-linked immunosorbent assay test; protein extraction and immunoblot analysis were performed on biopsied wounds. The first three most common agents involved in CVUs were Staphylococcus aureus (38·09%), Corynebacterium striatum (19·05%) and Pseudomonas aeruginosa (12·7%). In this study, we documented overall higher levels of MMP-1 and MMP-8 in patients with infected ulcers compared to those with uninfected ulcers that showed higher levels of MMP-2 and MMP-9. We also documented higher levels of interleukin (IL)-1, IL-6, IL8, vascular endothelial growth factor and tumour necrosis factor-alpha in patients with infected ulcers with respect to those with uninfected ulcers, documenting a possible association between infection, MMP activation, cytokine secretions and symptoms. The present results could represent the basis for further studies on drug use that mimic the action of tissue inhibitors of metalloproteinases in order to make infected CVU more manageable.
Introduction
Chronic venous disease (CVD), widespread in western countries (up to 57% in males and 77% in females), plays a major role in quality of life and work activities of affected patients (1–6). CVD is related to several complications that may be classified through Clinical-EtiologyAnatomy-Pathophysiology (CEAP) classification (7). Chronic venous ulcers (CVUs) are the most common type of wounds affecting the lower limbs (8–12) and they represent a common complication of CVD. CVUs represent the pathophysiological expression of an inflammatory process orchestrated by specific
and non-specific immune response; inflammatory cells provide growth factors and stimulate the deposition of matrix proteins
Key Messages • chronic venous ulceration (CVU) can result in several
complications, including the onset of infection, causing increasing pain and persistent ulceration • matrix metalloproteinases (MMPs) and inflammatory cytokines play an important role in both
© 2014 The Authors International Wound Journal © 2014 Medicalhelplines.com Inc and John Wiley & Sons Ltd doi: 10.1111/iwj.12225
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ECM, CVU, MMPs and cytokines Table 1 Characteristics of patients
pathophysiological mechanism of CVUs and infectious diseases; when bacteria begin replication and increase their metabolic activity, the resulting by-products, such as endotoxins and MMPs, negatively impact all phases of wound healing – moreover, inflammatory cytokines are important mediators of protein catabolism in infectious diseases • in our study, we documented higher levels of MMP-1 and MMP-8 and the cytokines, IL-1, IL-6, IL-8, VEGF and TNF-α, in patients with infected ulcers compared to those with uninfected ulcers • the present results could represent the basis for further studies on drugs that could lower the levels of MMPs and make infected CVUs more manageable
and phagocytose debris. CVUs are chronic skin lesions and may be complicated by infections (13,14); the risk of wound infection is increased when an imbalance occurs between local factors favouring bacterial growth and integrity of the immune defences. These lesions may become non-healing chronic wounds as long as the antigens from microorganisms remain. Several studies have shown that the inflammatory process characterising CVUs is associated with the presence of matrix metalloproteinase (MMP) activation (15–18). MMPs are related with extracellular matrix (ECM), a network of interlacing macromolecules that forms a supporting structure for vascular wall and skin integrity. ECM is dynamically maintained by the action of MMPs (which degrade ECM proteins) and their inhibitors (tissue inhibitors of MMPs, TIMPs) (19). Evidences have shown that MMPs play a central role in infectious diseases (20–23) also through ECM degradation, which favours the migration of immune cells from the bloodstream to the site of inflammation (24–26). The chronic exposure of CVUs to the environment, associated with the individual susceptibility of patients, predisposes the onset of infectious processes that worsen the prognosis for cure (27). In this study, we evaluated the expression of MMPs in infected ulcers and the levels of inflammatory cytokines. Materials and methods Study design
This study was approved by the Institutional Review Board–Independent Ethics Committee (IRB-IEC) of Interuniversity Center of Phlebolymphology – International Research and Educational Program in Clinical and Experimental Biotechnology – at University Magna Graecia of Catanzaro. Prior to the study, all participants were informed about the aim, procedures, risks and benefits of the study and a written informed consent was obtained. We conducted an open-label, parallel-group study between January 2011 and December 2012 in two Clinical Departments of Catanzaro and Naples. CVUs are included in stage 6 of CEAP classification (7). At the time of admission, patients’ medical history was recorded, and clinical examination, ulcer duplex ultrasonography and computed haemodynamic 2
Number of enrolled patients Male/female Age (years) Range Duplex ultrasound findings Superficial vein incompetence Perforating vein incompetence Superficial + perforating vein incompetence Ulcer features Ulcer area (range) Ulcer area (mean) Leucocytes: median/mm3 Concomitant conditions Diabetes Obesity Arterial hypertension COPD Malignancy Length of hospital stay (days ± SD)
Group I: patients with infected CVUs
Group II: patients with uninfected CVUs
63 27/36 59·4 ± 9 47–73
66 28/38 54·0 ± 8·6 45–70
11 (17·46%)
25 (37·88%)
25 (38·68%)
20 (30·30%)
27 (42·86%)
21 (31·81%)
2·5–19·9 cm2 12·7 cm2 14 600
2·1–17·8 cm2 10·5 cm2 11 000
14 10 21 6 12 9·4 ± 7·8
16 13 18 9 10 3·2 ± 9
CVU, chronic venous ulcer; COPD, chronic obstructive pulmonary disease.
mapping (28,29) were performed. Moreover, a blood sample was drawn at the time of admission for laboratory evaluation. The inclusion criteria were presence of infected venous ulcers, ankle-brachial pressure index (ABPI) >0·9 and, when associated with blood hypertension, a diastolic arterial pressure below 95 mmHg. The exclusion criteria were presence of arterial disease, connective tissue disorders including rheumatoid arthritis, blood disorders or cancer and ischaemia (ABPI < 0·9), smoking, systemic diseases, diabetes mellitus, use of any medications within the previous 7 days that could impair wound healing, and topical or systemic antimicrobial therapy. Patient characteristics are shown in Table 1. MMP evaluation in plasma and wound fluid
MMPs were measured in fluids of healing wounds and non-healing ulcers by enzyme-linked immunosorbent assay (ELISA), in agreement with previous studies (4,15–18,30–34). Protein extraction and immunoblot analysis Western blot was performed to estimate the expression of MMP-1, MMP-2, MMP-8 and MMP-9 in tissues, in agreement with previous studies (4,15–18,30,32–34). Wounds were subsequently biopsied (T = 1) under 1% lidocaine local anaesthesia and with full sterile precautions. The biopsy was made at a point equidistant from the centre and edge of the ulcer. Our experience with biopsies in this
© 2014 The Authors International Wound Journal © 2014 Medicalhelplines.com Inc and John Wiley & Sons Ltd
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patient population indicates that it is well tolerated by the subject and does not influence healing outcomes in venous ulcers. One biopsy was immediately placed into a sterile collection container and sent for quantitative (microbiology) culture. In both group I and group II, the biopsies obtained at the time of wound bed preparation (T = 1) were used for Western blot analysis in 2 ml of tissue protein extraction reagent (25 mM Bicine and 150 mM sodium chloride, pH = 7·6; Thermo Scientific, Cambridge, UK). Protein concentrations were determined and lysates were stored at 80◦ C. For Western blot, protein extracts were separated on a 12·5% sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes as previously described. Immunoblotting was performed using anti-MMP-1, anti-MMP-2, anti-MMP-8 and anti-MMP-9 monoclonal antibodies and expressed as arbitrary units. All experiments were performed in triplicate.
Biopsy
Biopsies were obtained under local anaesthesia with 2% lidocaine from the border area of the ulcer and from similar anatomical site of non-infected subjects. In order to eliminate bacterial infection, swabs for microbiology studies were obtained from all cases at the same time when biopsy was taken. The obtained tissue measured 10 mm length × 5 mm width, comprising the ulcer edge and surrounding skin. Each biopsy was divided into two halves: one was fixed in formaldehyde buffer with 10% glycine, while the other half was kept in RNAlater (Qiagen, Duesseldorf, Germany). The biopsies were embedded in paraffin, cut into 4-mm sections and stained with picrosirius. Collagen fibres were quantified under a microscope at 160× magnification using a millimetre ruler on the eyepiece in a 10-mm field. The thickness of each collagen fibre present in this field was measured. The quantity of collagen fibres was determined as the sum of the fibre thicknesses divided by 10 mm. For primary cell culture, the tissue was kept in Dulbecco’s modified Eagle’s medium and HAM F-12 for no longer than 2 hours. Biopsies were fixed for 24 hours and embedded in paraffin.
Immunohistochemical analysis
Sections of 4-mm thick were mounted on silane-coated slides, deparaffinised and the endogenous peroxidase was quenched with 0·03% H2 O2 in absolute methanol. Then, the sections were washed and blocked with phosphate-buffered saline supplemented with 2% goat serum. Skin sections were incubated with one of the following antibodies for 18 hours, anti-LL-37, anti-HBD-1, anti-HBD-2, anti-HBD-3, anti-HBD4 or anti-HNP-1, and then washed and incubated for 2 hours with anti-goat or anti-rabbit IgG biotin-labelled antibody. Bound antibodies were detected with avidin–biotin peroxidase and counterstained with haematoxylin. The whole biopsy area was taken for quantification. At 100× magnification, all negative or positive cells from the epidermis were counted using an image analyser.
ECM, CVU, MMPs and cytokines
The slides were counterstained with Harris haematoxylin. For all markers, the sections were analysed under a microscope at 400× magnification and the quantification was performed as the mean of the sum of labelled cells in each analysed field divided by the number of fields. The same procedure was used in the dermis. Cytokines analysis
An ELISA Kit (Human ELISA System, Amersham Pharmacia Biotech, Buckinghamshire, UK) was used to determine the concentration of interleukin (IL)-1, IL-6, IL-8, vascular endothelial growth factor (VEGF) and tumour necrosis factoralpha (TNF-α) in both plasma and wound fluid, in agreement with previous studies (15,18) Statistical analysis
Data analysis was performed using SigmaStats 3.5 (STACON, Witzenhausen, Germany). Kruskal–Wallis analysis of variance (ANOVA) was performed for all statistical comparisons between groups, and the Mann–Whitney U -test was performed for individual comparisons. All data are expressed as mean ± standard error of the mean (SEM) and P < 0·05 was considered significant. Results Patients
Enrolled patients were randomised into two groups. Group I comprised 63 patients (36 females and 27 males with a median age of 68·7 years) with infected CVUs, evidence of venous reflux at duplex scanning, and group II (control group) comprised 66 patients (38 females and 28 males with a median age of 61·2 years) with non-infected venous ulcers. Microbiological agents involved in CVUs
The most common agents involved in CVUs were Staphylococcus aureus (38·09%), Corynebacterium striatum (19·05%) and Pseudomonas aeruginosa (12·7%); remaining bacterial strains were as follows: Escherichia coli (3·17%), Staphylococcus epidermidis (4·76%), Alcaligenes faecalis (1·59%), Stenotrophomonas maltophilia (3·17%), Enterococcus faecium (4·76%), Streptococcus pyogenes (6·35%), Acinebacterium haemoliticus (3·17%), Providencia rettgeri (1·59%) and Staphylococcus simulans (1·59%) (Table 2). MMP values in plasma and tissues
ELISA test and Western blot analysis documented higher levels of MMP-1 and MMP-8 in group I, and MMP-2 and MMP-9 in group II (Figures 1 and 2). Dosage of inflammatory cytokines
ELISA test revealed higher levels of IL-1, IL-6, IL-8, VEGF and TNF-α in plasma and wound fluid of patients with infected CVU (group I) compared to those with uninfected CVU (group II) (Figure 3).
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Staphylococcus aureus Corinebacterium striatum Pseudomonas aeruginosa Escherichia coli Staphylococcus epidermidis Alcaligenes faecalis Stenotrophomonas maltophilia Enterococcus faecium Streptococcus pyogenes Acinebacterium haemoliticus Providencia rettgeri Staphylococcus simulans
MMPs plasma levels (ng/ml)
A
**
24 (38·09) 12 (19·05) 8 (12·7) 2 (3·17) 3 (4·76) 1 (1·59) 2 (3·17) 3 (4·76) 4 (6·35) 2 (3·17) 1 (1·59) 1 (1·59)
group I
group II
**
30 25 20
group I
group II
**
100 80 60 40 20 0
B
1200 1000
MMP-1 **
MMP-8 group I
group II
800 600 **
400 200 0
MMP-2
10
0
1200
MMP-1 **
MMP-8 group I
MMP-9
Figure 2 Tissue levels of matrix metalloproteinases (MMP)-1 and MMP8 (A) and MMP-2 and MMP-9 (B) measured through Western blot in enrolled patients at the time of admission. Group I: patients with infected ulcers; group II: patients with uninfected ulcers. **P < 0·01.
group II
1200
**
group I
group II
1000
1000 800
Plasma levels (pg/mL)
MMPs plasma levels (ng/ml)
**
120
15
5
B
MMPs tissues expression (Arbitrary Units)
N (%)
Species name
A
MMPs plasma levels (ng/mL)
Table 2 Percentage distribution of bacterial species in the samples of enrolled patients
600 **
400 200
**
**
**
800 600 400 200
0
MMP-2
MMP-9
0
Figure 1 Plasma levels of matrix metalloproteinases (MMP)-1 and MMP-8 (A) and MMP-2 and MMP-9 (B) measured through enzymelinked immunosorbent assay (ELISA) in enrolled patients at the time of admission. Group I: patients with infected ulcers; group II: patients with uninfected ulcers. **P < 0·01. Kruskal–Wallis analysis of variance (ANOVA) test was performed to compare plasma MMP-8 values between two groups.
Discussion
In this study, we evaluated the expression of MMPs and cytokines in patients with infected and non-infected CVUs. Wound healing progresses through several phases: (i) haemostasis; (ii) inflammation (removal of debris, control of infection and clearance of inflammation); (iii) proliferation 4
VEGF
TNF-alpha
IL-6
IL-8
Figure 3 Cytokine plasma levels of vascular endothelial growth factor (VEGF), tumour necrosis factor-alpha (TNF-α), interleukin (IL)-1, IL-6 and IL-8 measured through enzyme-linked immunosorbent assay (ELISA) in enrolled patients at the time of admission. Group I: patients with infected ulcers; group II: patients with uninfected ulcers. **P < 0·01.
(angiogenesis, deposition of granulation tissue and contraction); and (iv) remodelling (remodelling of the connective tissue matrix, and maturation) (35). The normal wound healing process is a complex physiological event that depends on interactions between epidermal keratinocytes, dermal fibroblasts, Langerhans cells, endothelial cells and fibroblasts, and is coordinated via complex cell–cell and cell–matrix
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interactions (36). CVUs are lesions characterised by prolonged inflammation, a defective wound matrix and failure of reepithelialisation; these factors facilitate the colonisation by major pathogens, particularly Gram-positive and Gram-negative bacteria. This study showed a high prevalence of S. aureus infections, followed by P. aeruginosa and C. striatum. These bacterial species had evolved numerous mechanisms to evade host defence strategies employed by neutrophils, including the ability to modulate normal neutrophil turnover, a process critical to the resolution of acute inflammation. Gender, age (37), defence mechanisms (33), CVU severity [according to CEAP classification (7)] and hormonal and metabolic status of the host are among the factors influencing the vital activity of commensal. Wound infection advances in stages from contaminated, colonised, critically colonised to infected wounds. When bacteria begin replication and increase their metabolic activity, the resulting by-products, such as endotoxins and MMPs, negatively impact all phases of wound healing. MMPs play a central role in infectious diseases when the host immune system is challenged by an invading organism, facilitating the recruitment of leucocytes from the bloodstream; these migrate to the site of infection for eradication of the pathogen (by proteolysis of the basement membrane) and for modulating the inflammatory response (31). In this study, we documented higher levels of MMP-1 and MMP-8 in patients with infected ulcers compared to those with uninfected ulcers that showed higher levels of MMP-2 and MMP-9. These results document a different role of MMPs during the infection, and these results may be in agreement with other studies (34,38,39) that documented that MMP-1 is increased upon stimulation with inflammatory cytokines such as IL-1β, TNF-α and IL-6 showing a correlation between MMPs and cytokines. Inflammatory cytokines, such as TNF-α, IL-1 and IL-6, are important mediators of protein catabolism in infectious diseases (40–43) such as infected CVUs. Moreover, polymorphonuclear granulocytes produce MMP-8, a neutrophil collagenase, which is pivotal for initiation and consequent resolution of inflammation, presumably by altering chemotactic gradients (44). TNF-α and IL-1 play a central role in the regulation of collagen metabolism in fibroblasts; addition of recombinant TNF to cultured human fibroblasts significantly decreased both collagen gene transcription and peptide synthesis (45,46). TNF also modulates collagen metabolism by increasing the synthesis of collagenase, an enzyme that digests collagen (47). In this study, we documented higher levels of IL-1, IL-6, IL-8, VEGF and TNF-α in plasma and wound fluid of patients with infected ulcers compared to those with uninfected ulcers, documenting a possible association between infection, MMP activation, cytokine secretions and symptoms. Patients with severe or persistent infections are at particularly high risk for problems related to poor wound healing and these problems require either reoperation or prolonged medical care (48,49). Previously, in an experimental study, we documented that corticosteroid may be able to modulate the expression of TNF-α and inflammatory cytokines (50) in epithelial cells; therefore, the present results could represent the basis for further studies on drug use that mimic the action of TIMP-1,
ECM, CVU, MMPs and cytokines
a tissue inhibitor of MMPs (51); these could lower the levels of MMPs and make CVUs more manageable. Acknowledgements
This study received no funding. The authors declare that they have no competing interests. References 1. Robertson L, Evans C, Fowkes FGE. Epidemiology of chronic venous disease. Phlebology 2008;23:103–11. 2. Beebe-Dimmer JL, Pfeifer JR, Engle JS, Schottenfeld D. The epidemiology of chronic venous insufficiency and varicose veins. Ann Epidemiol 2005;15:175–84. 3. Serra R, Grande R, Buffone G, Costanzo G, Damiano R, de Franciscis S. Chronic venous disease is more aggressive in patients with varicocele. Acta Phlebol 2013;14:57–60. 4. Serra R, Buffone G, Costanzo G, Montemurro R, Scarcello E, Stillitano DM, Damiano R, de Franciscis S. Altered metalloproteinase-9 expression as the least common denominator between varicocele, inguinal hernia and chronic venous disorders. Ann Vasc Surg 2013, In press, pii: S0890-5096(13)00576-1. DOI: 10.1016/j.avsg.2013.07.026. 5. Serra R, Buffone G, de Franciscis A, Mastrangelo D, Molinari V, Montemurro R, de Franciscis S. A genetic study of chronic venous insufficiency. Ann Vasc Surg 2012;26:636–42. 6. Serra R, Buffone G, Costanzo G, Montemurro R, Perri P, Damiano R, de Franciscis S. Varicocele in younger as risk factor for inguinal hernia and for chronic venous disease in older: preliminary results of a prospective cohort study. Ann Vasc Surg 2013;27:329–31. 7. Ekl¨of B, Rutherford RB, Bergan JJ, Carpentier PH, Gloviczki P, Kistner RL, Meissner MH, Moneta GL, Myers K, Padberg FT, Perrin M, Ruckley CV, Smith PC, Wakefield TW, American Venous Forum International Ad Hoc Committee for Revision of the CEAP Classification. Revision of the CEAP classification for chronic venous disorders: consensus statement. J Vasc Surg 2004;40:1248–52. 8. Serra R, Buffone G, Molinari V, Montemurro R, Perri P, Stillitano DM, Amato B, de Franciscis S. Low molecular weight heparin improves healing of chronic venous ulcers especially in the elderly. Int Wound J 2013, In press. DOI: 10.1111/iwj.12071. 9. de Franciscis S, De Sarro G, Longo P, Buffone G, Molinari V, Stillitano DM, Gallelli L, Serra R. Hyperhomocysteinaemia and chronic venous ulcers. Int Wound J 2013, In press. DOI: 10.1111/iwj.12042. 10. Serra R, Buffone G, de Franciscis A, Mastrangelo D, Vitagliano T, Greco M, de Franciscis S. Skin grafting followed by low-molecularweight heparin long-term therapy in chronic venous leg ulcers. Ann Vasc Surg 2012;26:190–7. DOI: 10.1016/j.avsg.2011.04.008. 11. de Franciscis S, Grande R, Buffone G, Serra R. Chronic venous ulceration of the lower limbs and thrombosis. Acta Phlebol 2013, In press. 12. Gasbarro V, Amato B, Izzo M, Manfrini A, Buffone G, Grande R, Serra R, de Franciscis S. Quercetin and chronic venous ulceration of the lower limbs. Acta Phlebol 2013;14:61–5. 13. Edwards R, Harding KG. Bacteria and wound healing. Curr Opin Infect Dis 2004;17:91–6. 14. Debats IB, Booi D, Deutz NE, Buurman WA, Boeckx WD, van der Hulst RR. Infected chronic wounds show different local and systemic arginine conversion compared with acute wounds. J Surg Res 2006;134:205–14. 15. Serra R, Buffone G, Falcone D, Molinari V, Scaramuzzino M, Gallelli L, de Franciscis S. Chronic venous leg ulcers are associated with high levels of metalloproteinases-9 and neutrophil gelatinaseassociated lipocalin. Wound Repair Regen 2013;21:395–401. DOI: 10.1111/wrr.12035.
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