Differential Proteomic Analysis Distinguishes Tissue Repair Biomarker

Jul 28, 2010 - and Center for Biochemistry, University of Cologne, Cologne, Germany, Department of Dermatology, University. Medical Center Freiburg ...
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Differential Proteomic Analysis Distinguishes Tissue Repair Biomarker Signatures in Wound Exudates Obtained from Normal Healing and Chronic Wounds Sabine A. Eming,*,† Manuel Koch,‡,|,⊥ Andreas Krieger,† Bent Brachvogel,⊥ Sandra Kreft,⊥ Leena Bruckner-Tuderman,#,∇ Thomas Krieg,†,‡,§ John D. Shannon,O and Jay W. FoxO Department of Dermatology, Center for Molecular Medicine Cologne (CMMC), Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), Institute for Oral and Musculoskeletal Biology, and Center for Biochemistry, University of Cologne, Cologne, Germany, Department of Dermatology, University Medical Center Freiburg, Germany, Freiburg Institute of Advanced Studies, School of Life Sciences-LifeNet, Germany, and Department of Microbiology, University of Virginia, Charlottesville, Virginia Received May 14, 2010

Chronic wounds associated with vascular disease, diabetes mellitus, or aging are leading causes of morbidity in western countries and represent an unresolved clinical problem. The development of innovative strategies to promote tissue repair is therefore an important task that requires a more thorough analysis of the underlying molecular pathophysiology. We propose that the understanding of the complex biological events that control tissue repair or its failure largely benefits from a broad analytical approach as provided by novel proteomic methodologies. Here we present the first comparative proteome analysis of wound exudates obtained from normal healing or nonhealing (venous leg ulcer) human skin wounds. A total of 149 proteins were identified with high confidence. A minority of proteins was exclusively present in exudate of the healing wound (23 proteins) or the nonhealing wound (26 proteins). Of particular interest was the differential distribution of specific proteins among the two different healing phenotypes. Whereas in the exudate obtained from the healing wound mediators characteristic for tissue formation were abundantly present, in the exudate obtained from the nonhealing wound numerous mediators characteristic for a persistent inflammatory and tissue destructive response were identified. Furthermore, the study also revealed interesting results regarding the identification of new proteins with yet unknown functions in skin repair. This analysis therefore represents an important basis for the search for potential biomarkers, which give rise to a better understanding and monitoring of disease progression in chronic wounds. Keywords: Wound healing • chronic ulcers • biomarker • wound exudate • inflammation

Introduction Skin injury induces a complex network of signaling systems including growth factors, their receptors, extracellular matrix molecules, and different classes of proteases as well as their inhibitors.1 The stringently regulated interaction of these mediators directs the restoration of the epidermis by epithelialization and of dermal structures by granulation tissue formation and matrix deposition. Numerous experimental studies have identified crucial growth factors, their receptors, extracellular matrix molecules, and downstream signaling components directing a suc* To whom correspondence should be addressed. Phone: ++49-2214783196. Fax: ++49-221-4200988. E-mail: [email protected]. † Department of Dermatology, University of Cologne. ‡ CMMC, University of Cologne. § CECAD, University of Cologne. | Institute for Oral and Musculoskeletal Biology, University of Cologne. ⊥ Center for Biochemistry, University of Cologne. # University Medical Center Freiburg. ∇ Freiburg Institute of Advanced Studies. O University of Virginia.

4758 Journal of Proteome Research 2010, 9, 4758–4766 Published on Web 07/28/2010

cessful repair response, and based on these studies much insight has been gained into human wound physiology.2,3 In contrast, mechanisms leading to impairment of the wound healing response are poorly understood, and currently no efficient therapy is available for chronic wounds.4 For most patients an underlying systemic disease interferes with local tissue repair mechanisms, and the result is a chronic wound that does not heal as expected. Common systemic diseases and conditions underlying impaired wound healing include venous insufficiency, diabetes, aging, and skin fragility.5 Chronic skin wounds cause morbidity and mortality and represent a significant clinical problem as well as an enormous financial burden to health care systems.6 A better understanding of the functional mechanisms leading to the pathology of impaired healing complications on the molecular level is fundamental to monitor disease progression and to offer new perspectives for the development of effective therapies for wound healing disorders. In this study we apply a broad analytical approach as provided by novel proteomic methodologies to understand the 10.1021/pr100456d

 2010 American Chemical Society

Comparative Proteome Analysis of Wound Exudates complex biological processes that represent efficient tissue repair or its failure. In mammals to date no single factor essential has been identified, and it is speculated that the healing or nonhealing phenotype is associated with manifold relevant biomarkers. The identification of biomarkers that predict a healing response would represent a milestone in the understanding of the tissue repair pathology and potentially point to novel therapies for chronic wounds. Wound exudate is a complex composition, containing soluble factors released into the wound microenvironment, which in turn may influence cellular functions at the wound site. Therefore, the exudate is a liquid biopsy that reflects the metabolic condition of the wound and has been proven to be useful in identifying factors involved in skin repair or its failure.7–10 Proteomics provides highly useful methodologies to analyze complex protein mixtures and consequently can be used for the comparative analysis of a variety of fluids in different conditions. Proteomic analyses have been performed in various body fluids including wound fluid11–14 and suction blister,15 and recently a systematic database for human fluid proteome research has been developed.16 Thus the possibility of using proteomic tools to characterize the wound environment through the analysis of exudate is a highly promising approach to gain a better understanding of the complex mechanisms underlying pathologic phenomena. Here we present the first comparative proteome analysis of wound exudates obtained from one representative normal and one representative nonhealing (chronic venous leg ulcer) human skin wound. This investigation revealed three major findings. First, we identified several known mediators involved in skin repair. Second, the study yielded surprising results regarding new proteins with yet unknown functions in wound healing. Third, several proteins were identified that clearly distinguish a healing from a nonhealing skin microenvironment. Whereas mediators that promote tissue growth and protect from inflammation-mediated tissue damage characterize the healing phenotype, products of a chronic inflammatory response, including leukocyte proteases, pro-inflammatory mediators, as well as cellular components indicating cellular damage predominate the nonhealing state. We propose our analysis as a discovery type of experiment using limited patient samples to seek for potential biomarkers. Many potential biomarkers were identified, and three, lactoferrin, annexinA1 (ANXA1), and S100A9, as a proof of concept, were validated via ELISA, dot blot analysis, and immunohistochemical staining, thereby demonstrating that this is a feasible first step in biomarker discovery. Therefore, our study contributes to an enhanced understanding of a successful healing response or its failure and could be used as monitor for disease activity.

Experimental Section Collection of Wound Exudate. Wound exudate was obtained from patients presenting with ulcera crura due to venous insufficiency (n ) 19; mean age 67 years, persistence of ulcus for 2-3 years without any tendency to heal) or from patients with normal healing acute cutaneous wounds (n ) 9, excision wounds of the lower leg 8-10 days post surgery, awaiting wound closure by secondary intention; mean age 65 years). For this purpose the wound was covered with a semipermeable polyurethane film (Hyalofilm, Hartmann, Heidelberg, Germany) for a maximum of 8 h. Following collection (usually 1 mL was obtained), fluids were centrifuged (10 min, 13,000 × g, 4 °C) to remove insoluble material, and supernatants were frozen at

research articles -80 °C until use. The study was approved from the local ethics committee and informed consent of patients was received. Mass Spectrometry Data Analysis and Presentation. For mass spectrometry analysis one representative exudate sample obtained from the nonhealing wounds and one sample obtained from the healing wounds were analyzed. Lyophilized wound exudate samples were resuspended in water, and protein quantification was performed using a NanoOrange protein kit (Invitrogen). Sixty micrograms of protein was acetone precipitated, resuspended in Laemmli buffer, applied to a 4-20% precast electrophoresis gel (Bio-Rad), and stained with Coomassie Blue. Gel lanes were cut into 10 equal size slices. Gel slices were destained for 2 h, and the proteins were reduced (10 mM DTT) and alkylated (50 mM iodoacetamide) at room temperature. Gel slices were washed with 100 mM ammonium bicarbonate, dehydrated with acetonitrile, and dried in a speed vac. Hydration of the slices was performed with a solution of Promega modified trypsin (20 ng/mL) in 50 mM ammonium bicarbonate for 30 min on ice. Excess trypsin solution was removed, and the digestion was carried on for an additional 18 h at 37 °C. Tryptic peptides were twice extracted from gel slices with 30 µL of a 50% acetonitrile/5% formic acid solution. The combined extracts were dried to a volume of 15 µL for mass spectrometric analysis. LC-MS/MS was performed using a Thermo Electron LTQ ion-trap mass spectrometer. Analytical columns were fabricated in-house by packing 7.5 cm Jupiter 10 µm C18 packing material (Phenomenex, Torrance, CA) into a 25 cm length of 360 × 75 µm fused silica (Polymicro Technologies, Phoenix, AZ) behind a bottleneck. Samples were loaded directly onto these columns for the C18 analytical runs. In-gel digests (50% of each sample) were injected into the mass spectrometer at 300 nL/min. Peptides were eluted from the C18 column using an acetonitrile/0.1 M acetic acid gradient (2-90% acetonitrile). The instrument was programmed to acquire a cycle of one mass spectrum followed by MS/MS on the 10 most abundant ions in a data-dependent mode. After MS/MS, fragmentation was carried out on a particular parent ion and the m/z was placed on an exclusion list for 2 min to enable greater dynamic range and prevent repeat analysis of the same ions. The electrospray voltage was set to 2.5 kV, and the capillary temperature was 230 °C. The mass spectra were extracted and analyzed utilizing Bioworks Sequest 3.11 software. Searches were performed against a mouse IPI nonredundant database (http://www.ebi.ac.uk/IPI/). Spectra generated on the LTQ were searched using 1.5 Da parent tolerance and 1 Da fragment tolerance. All hits were required to be fully tryptic. The results from the searches were exported to Scaffold (version 2.2.03, Proteome Software Inc., Portland, OR). Scaffold was used to validate MS/MS based peptide and protein identifications and to visualize multiple data sets in a comprehensive manner. Confidence of protein identification in Scaffold is displayed as a Probability Legend with green coloration indicative of >95% confidence and yellow as 80-94% confidence. Relative quantification of proteins was accomplished by summing all data from the 10 gel slices for a particular sample in Scaffold and then displaying the Quantitative Value from the program. This number gives an average total of nongrouped spectral counts for a protein divided by the total nongrouping spectral counts for the 10 mass spectral runs from the gels slices from each lane (http://www.proteomesoftware.com/). This format of presentation allows for a relative quantitative comparison between a specific protein from different samples and to a certain degree gives some Journal of Proteome Research • Vol. 9, No. 9, 2010 4759

research articles measure of relative abundance between proteins generated from the mass spectrometric analysis of the 10 gel slices for a particular exudate sample. Lactotransferrin Quantification. As an indication that proteomic analysis of the two patient samples with different wound healing properties could be representative of larger patient populations, the concentration of lactotransferrin in wound exudates was determined in 14 different samples obtained from patients with nonhealing wounds and 5 samples obtained from patients with healing wounds, using a commercially available ELISA that detects human Lactotransferrin (AssayMax Human Lactotransferrin ELISA, Assaypro, St. Charles, MO). Lactotransferrin levels were normalized to the total protein concentration, which was determined by the Bradford procedure (Bio-Rad Protein Assay, Bio-Rad, Mu ¨ nchen, Germany). S100A9 Quantification. The concentration of S100A9 in wound exudates was determined in 14 different samples obtained from patients with nonhealing wounds and 5 samples obtained from patients with healing wounds, using a commercially available ELISA that detects human S100A9 (Uscn Life Science Inc., Wuhan, China). S1009A levels were normalized to the total protein concentration, which was determined by the Bradford procedure (Bio-Rad Protein Assay, Bio-Rad, Mu ¨ nchen, Germany). Immunohistochemistry. To process tissue sections for the immunodetection of Lactotransferrin, S100A9 and CD11b 5 µm cryosections were fixed in 4% PFA, rinsed, and then blocked with 10% FCS/PBS to reduce nonspecific antibody binding. Sections were incubated (1 h, RT) with polyclonal rabbit antibody against lactotransferrin (Dianova, Augst, Switzerland), a monoclonal antibody against human S100A9 (BMA Biomedicals, Augst, Switzerland), and with polyclonal rat antibody against CD11b (Miltenyi, Bergisch Gladbach, Germany). Bound primary antibodies were detected using an Alexa 488-conjugated antibody against mouse IgG and Alexa 568-conjugated antibodies against goat or rabbit IgG (Molecular Probes, Cambridge, U.K.). DAPI was used for counterstaining. Specificity of primary antibodies was demonstrated by an irrelevant isotype-matched rabbit or rat antibody. Annexin Detection Assay. Excudates (venus leg ulcer, healing wound, serum) were spotted onto a nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany). After blocking in Trisbuffered saline (TBS), 0.1% Tween-20, 2 mg/mL fatty acid-free bovine serum albumin (BSA), annexins were detected by adding rabbit-antiserum against ANXA1 (Zymed, San Francisco, CA), ANXA3 (unpublished), and ANXA5 (Hyphen Biomed, Neuvillesur-Oise, France) and goat-antiserum against ANXA2 (Santa Cruz Biotechnology, Santa Cruz, CA) in blocking solution. The primary antibody was detected with a swine antirabbit/goat IgG labeled with horseradish peroxidase (DAKO, Glostrup, Denmark) and visualized by chemoluminescence using standard procedures. The integrated density of the dot plots were analyzed using imageJ software. The background signal of the dot plots was subtracted prior to analysis.

Results Proteomic Analysis of Wound Exudates from Normal Healing and Nonhealing Wounds. A series of patient samples was analyzed by SDS-PAGE, and for mass spectrometric analysis two samples, which appeared to be representative of the healing and nonhealing groups (Figure 1) were analyzed; 123 and 126 proteins were identified with over 99% confidence (as per the Scaffold algorithm, at least two unique peptides per 4760

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Figure 1. 1D SDS-PAGE of diverse wound exudates. Exudates from patients with either normal healing acute cutaneous wounds (A-G; A and E 3 days post surgery; B, C, and F 7 days post surgery; D and G 15 days post surgery) or samples from patients with a chronic venous leg ulcer (H-K) were electrophoresed on a 4-20% gradient gel followed by staining with Coomassie Blue. The box indicates the samples that were selected for mass spectrometric analysis and grid depicts the gel slice positions for mass spectrometry analysis processing. Note that in K the presence of full length serum albumin is reduced due to the increased proteolysis in the wound exudates from nonhealing wounds. Table 1. Extracellular Matrix Proteins

protein) for the healing and nonhealing wounds, respectively. Of these proteins, 23 and 26 were unique to the data sets for the healing and nonhealing wounds, respectively, and are suggestive that they may be biomarkers elucidated in the exudates relevant to the status of the wound. Most of the proteins were present in both exudates obtained from healing as well as nonhealing wounds. Of particular interest was the distribution of specific proteins among the two different healing conditions. The most abundant proteins identified on the basis of their quantitative value were sorted into 4 categories on the basis of their proposed key function in tissue repair or its failure: extracellular matrix molecules (Table 1); proteinases (Table 2); different groups of inhibitors (Table 3); and immune modulatory mediators (Table 4). Values marked in gray in the tables demonstrate a >2-fold increase compared to the other exudate. The full data set is found in the Supporting Information. Differential Detection of Extracellular Matrix Molecules in Exudate of Healing and Nonhealing Wounds. Of the 15 most abundant extracellular matrix molecules identified, olfactomedin-4 was the only protein exclusively identified in exudate of the nonhealing wound (Table 1). Olfactomedin-4 is a member of the olfactomedin family of proteins, with yet

Comparative Proteome Analysis of Wound Exudates Table 2. Proteases

Table 3. Different Groups of Inhibitors

Table 4. Immunmodlulators

unknown function in tissue repair. The 5 most abundant extracellular matrix molecules exclusively detected in exudate of healing wounds, include collagens (Col1A1, Col1A2, Col3A1), heparin sulfate proteoglycan 2, matrix components regulating collagen fibril assembly (COMP), and basement membrane (fibulin-1) organization. Furthermore, tetranectin, a plasminogen-binding C-type lectin, revealed a 6-fold increase in healing wounds versus nonhealing wounds. Extracellular matrix components that are hallmarks of the provisional wound matrix were identified with higher quantitative values in nonhealing wounds compared to healing wounds. Those include the gamma and the beta chain of fibrinogen as well as vitronectin, fibronectin, lumican, and R2-HS-glycoprotein. Differential Detection of Proteases in Exudate of Healing and Nonhealing Wounds. Of the 7 most abundant proteases detected, MMP9, neutrophil elastase, and proteinase 3 were exclusively detected in exudate obtained from the

research articles nonhealing wound (Table 2). MMP9 represented the protease with the highest fold increase over those in exudate derived from healing wounds, with a quantitative value increased 22fold. Thrombin and Kallikrein B were limited to the exudate derived from the healing wound. These results indicate a dysbalance of leukocyte-derived proteases among the two exudates with a clear shift of increased proteases presence in the nonhealing microenvironment. Differential Detection of Protease Inhibitors in Exudate of Healing and Nonhealing Wounds. The most abundant identified protease inhibitors belong to the family of serine protease inhibitors (SERPINs) (Table 3). Among those, SERPINA1, SERPINA3, and SERPING1 revealed the highest quantitative value, and all 3 proteins were increased in exudates derived of healing wounds versus nonhealing wounds. SERPIND1 and SERPINB4 were exclusively detected in exudate derived from the nonhealing wound; however, both proteins were low in quantity. SERPINF1 protein level was 3.4-fold increased in exudate obtained from the healing versus the nonhealing wound. SERPINF1 has been shown to act as strong inhibitor of angiogenesis, and to date, no protease-inhibitory activity for SERPINF1 has been identified. Because of its structural homology to SERPINS, it has been listed in Table 3. Cystatins represent an additional family of protease inhibitors, of which Cystatin-C and -M were exclusively detected in exudate of the healing wound and Cystatin-A was limited to the exudate of the nonhealing wound. These results indicate an increased presence of protease inhibitors in the healing microenvironment. Differential Detection of Immune Modulators in Exudate of Healing and Nonhealing Wounds. Numerous proteins in the category of immune modulatory mediators were identified in exudate of nonhealing wounds and to a lesser extend in healing wounds (Table 4). Most of these proteins have already been associated with pro-inflammatory disease conditions in skin or other organs. Among all 149 proteins identified in the proteome analysis, lactotransferrin was identified as the protein having the highest abundance in the exudate obtained from nonhealing wounds over those obtained from healing wounds. This observation was confirmed in a larger cohort of exudate samples. Mean lactotransferrin concentration in exudates obtained from nonhealing wounds (n ) 14) was 10-fold increased over that in exudates obtained from normal healing wounds (n ) 5) (p < 0.001) (Figure 2A). Furthermore, local synthesis of lactotransferrin at the wound site in both healing and nonhealing wounds was revealed by its significantly lower concentration in blood serum (n ) 7) obtained from patients with normal healing or nonhealing wounds. To assess the distribution of lactotransferrin protein within the wound tissue, we performed immunohistochemistry studies on cryosections obtained from normal healing and nonhealing human skin wounds caused by venous insufficiency. Whereas in nonwounded normal skin lactotransferrin was not detectable (data not shown), a few cells within the granulation tissue of the healing wound 8 days post injury stained positive (Figure 2B). In contrast, in the nonhealing ulcer numerous cells throughout the dermal tissue stained strongly positive for lactotransferrin. Double staining with CD11b identified myeloid cells as the primary cell types expressing lactotransferrin in healing and nonhealing wounds (Figure 2B). Furthermore, among the category of immune modulators, several members of the S100 protein family were identified to be increased in the exudate obtained from the nonhealing Journal of Proteome Research • Vol. 9, No. 9, 2010 4761

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Figure 2. Lactrotransferrin is increased in nonhealing versus healing wounds. (A) Quantification of lactotransferrin in exudates obtained from healing wounds (n ) 5), venous leg ulcers (n ) 14), and blood serum (n ) 7); each dot represents the lactotransferrin concentration in wound exudate or blood serum obtained from a different patient; healing vs nonhealing p < 0.001. (B) Immunohistochemistry of lactotransferrin in granulation tissue of a healing (day 8 post injury) or nonhealing wound; in the nonhealing wound high numbers of myeloid cells (CD11b, green) stained positive for lactotransferrin (red), arrows indicate double positive stained cells (yellow), (blue cells, DAPI stain); scale bar 50 mM.

versus the healing wound (Table 4). As revealed by ELISA in a larger cohort of patient samples the mean concentration of S100A9 was significantly increased in exudates obtained from nonhealing wounds (n ) 14) versus healing wounds (n ) 6, p ) 0.038) (Figure 3A). In blood serum of these patients no S100A9 could be detected. As demonstrated by immunohistochemical staining in nonhealing wounds a strong signal for S100A9 was detected in the suprabasal layers of the hyperproliferative epithelial wound edge as well as in the papillary dermis and individual dermal cells within the granulation tissue, which only to some extent revealed double staining with CD11b (Figure 3B). In normal healing wounds S100A9 staining was limited to suprabasal keratinocytes in the vicinity to the wound edge, dermal staining was minimal (Figure 3B). In addition, based in the proteome analysis also the abundance of annexin A1 was increased in the exudate derived from the nonhealing versus the healing wound (Table 4). To corroborate this finding the presence of this protein family was validated by dot plot analysis in a larger cohort of patient samples. Exudates of nonhealing wounds (n ) 19), healing wounds (n ) 9), and blood serum (n ) 7) were immobilized on nitrocellulose membranes and annexin A1 (ANXA1), annexinA2 (ANXA2), annexin A3 (ANXA3), and annexin A5 (ANXA5) were detected by annexin specific antibodies (Figure 4A). A strong reaction product was detected for ANXA1 in the majority of exudates of nonhealing wounds (19 positive samples out of 4762

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Figure 3. S100A9 is increased in nonhealing versus healing wounds. (A) Quantification of S100A9 in exudates obtained from healing wounds (n ) 6) and venous leg ulcers (n ) 14); each dot represents the S100A9 concentration in wound exudate obtained from a different patient; healing vs nonhealing p ) 0.038. (B) Immunohistochemistry of S100A9 (red stain) in wound tissue of a healing (day 8 post injury) or nonhealing wound; in the healing wound S100A9 staining was limited to suprabasal keratinocytes at the hyperproliferative wound edge; in nonhealing wounds an intensive signal for S100A9 was detectd in the papillary dermis, suprabasal layers of the hyperproliferative wound edge, and to some extent CD11b positive cells (CD11b, green) stained also for S100A9. Arrows indicate double positive stained cells (yellow) (bluecells,DAPIstain),anddottedlineindicatestheepidermal-dermal junction.

19 total samples). In contrast, only a weak signal for ANXA1 was detected in most of the exudates obtained from healing wounds (6 weakly positive samples out of 9 total samples), and no signal was detected in any of the blood serum samples. ANXA2, ANXA3, and ANXA5 could not be detected at significant levels in any of the analyzed samples. Relative quantification of ANXA1 levels confirmed the significant upregulation of ANXA1 in the exudates of nonhealing wounds (Figure 4B, p e 0.01). Except for dermicidin, all antioxidant and antimicrobial defense proteins that were detected in the proteome analysis were exclusively present in exudate derived from the nonhealing wound. Among those, Myeloperoxidase and Lipocalin represented the most abundant proteins. Dermicidin was exclusively present in exudate derived from the healing wound. Numerous proteins of the complement factor family were identified in exudates of both healing conditions. Exclusive

Comparative Proteome Analysis of Wound Exudates

research articles sent statistical significance in itself. However, we do believe that valuable leads can be gained from this approach that can rapidly be verified by additional techniques such as ELISA and dot plot analysis to query statistically significant populations of samples. In the current study we have demonstrated this with lactrotransferrin, ANXA1 and S1009A. Of particular interest was the differential distribution of specific proteins among the two different healing conditions. Whereas mediators that promote tissue growth and protect from inflammation mediated tissue damage characterize the healing phenotype, products of a chronic inflammatory response, including leukocyte proteases and pro-inflammatory mediators, predominate the nonhealing state.

Figure 4. AnxA1 is increased in nonhealing versus healing wounds and serum. (A) Exudates obtained from venous leg ulcer (1-19) and blood serum (20-26) were spotted onto a nitrocellulose membrane, and annexins were detected with specific antibodies. Recombinant annexins were used as control (Co). (B) Relative quantification of AnxA1 in exudates of venous leg ulcer (1, 4-9, 11-19), blood serum (20, 21, 24-26), and healing wounds (1-9). The evaluation of the mean density values for each plot is illustrated, and the total mean value is given (line). Significance was tested by Student’s t test: **P e 0.01.

detection of Complement factor D and Complement component 7 in exudate of the healing wound as well as at least a 2-fold increase of Complement component 5 and 8 in exudate derived from the healing wound versus the nonhealing wound indicate a slight increase of proteins of the complement family in the healing microenvironment.

Discussion Here we present the first comparative proteome analysis of wound exudates obtained from a representative normal healing versus representative nonhealing (venous leg ulcer) human skin wound. This investigation revealed three major findings. First, we identified several known mediators involved in skin repair. Second, several proteins were identified that clearly distinguish a healing from a nonhealing skin microenvironment. Third, there are interesting results regarding novel proteins with yet unknown functions in wound healing. Therefore, we propose our analysis as basis for the search for surrogate biomarkers that offer new perspectives for a better understanding and prediction of a successful healing response or its failure. Proteomic analysis of biological fluid can be problematic given the issues of low analyte concentration, dilution, and distance from the lesion of interest.13,17,18 The approach we outline in this investigation appears to circumvent some of these issues particularly with regard to being proximal to the lesion (the wound). As mentioned we believe this approach provides a “proteomic window” on the status of the wound. This is validated by the fact that several notable factors associated with the nonhealing status were identified in this study including MMP 9,9,10,19 S100 proteins,20 and neutrophil elastase.8 We recognize that the analysis of only two representative wound exudates by mass spectrometry does not repre-

In the exudate obtained from the healing wound several extracellular matrix proteins were identified with significant increase over those present in exudate obtained from the nonhealing wound. Remarkably, all proteins identified of the collagen family were exclusively detected in the exudate derived from the healing wound. Collagen I, as the most abundant interstitial collagen in the skin, revealed the highest quantitative value, next to collagen III. In diverse experimental model systems as well as in humans, collagen III is well established as the hallmark collagen upregulated during the phase of granulation tissue formation in skin repair.21 These observations link the presence of collagen I and III to a physiological healing response and lead to the intriguing hypothesis that deficiency in collagen I and/or III may reflect an impaired healing state. Furthermore, among the category of extracellular matrix molecules, tetranectin represented a 6-fold increase in healing wounds versus nonhealing wounds. Tetranectin represents a plasminogen-binding C-type lectin, which enhances plasminogen activation.22 Tetranectin has been originally purified from human serum; however, to our knowledge its presence in human wounds has not been described so far. Recent studies reported that mice deficient in tetranectin display severe wound healing defects.23 However, in this study the functional mechanisms of tetranectin-mediated wound healing events were not further analyzed and remain elusive. Our findings support a role for tetranectin in skin wound healing and we propose further studies on the molecular level to identify its role in tissue repair or its failure in the murine but also human system. Fibronectin, fibrinogen, vitronectin, olfactomedin-4, and a2HS-glycoprotein were identified as the most abundant extracellular matrix molecules present in exudate derived from the nonhealing wound. These proteins are all classical components of the provisional wound matrix which characterizes the early phase of granulation tissue formation. On the basis of the fact that these matrix molecules predominate the chronic wound environment, we speculate that their presence reflects a nonproductive and frustrating wound healing response that is trapped in the early phase of the tissue repair response. Furthermore, these findings are in line with the lack of collagen I and III in the nonhealing wound, two biomarkers indicating the progression of the repair response into a phase of granulation tissue maturation (see above). Olfactomedin-4 represented the only extracellular matrix molecule exclusively detected in exudate of the nonhealing wound. Interestingly, the olfactomedin-family proteins have only recently been identified, and their biological roles remain largely elusive. A proposed function in tissue remodeling is based on recent reports describing their indispensable role in Journal of Proteome Research • Vol. 9, No. 9, 2010 4763

research articles embryonic axial pattern formation, neural development, and cell attachment in Xenopus.24,25 A cardinal feature of chronic wounds is a persistent inflammatory response, which is morphologically characterized by a dense cellular infiltrate consisting of neutrophils and tissue macrophages.26 Unbalanced proteolytic activity, which overwhelms local tissue protective mechanisms, has been demonstrated by many laboratories including our own, and uncontrolled proteolysis is considered a major consequence of the persistent inflammatory response at the chronic wound site7,8,10,19,26–29 Factors crucial for repair have been shown to be targets of proteases present in the chronic wound environment, and their proteolysis is considered to play an important pathogenic role.7,8 Our findings in the present analysis support this pathogenic concept. Among the category of proteases, several classes of proteases were identified, in particular serine proteinases and MMPs. To our knowledge, all of the most abundant proteases identified have already been reported in the literature to play important roles in skin repair.7,8,10,19,26–29 Furthermore, also the tendency of these proteases to be increased in the chronic versus the healing microenvironment, has been reported. In particular, the high quantitative values for MMP9,10,16,25 leukocyte elastase,9,27 and proteinase 326 in wound exudate of venous ulcers has been reported earlier. Most of these proteases are synthesized by polymorphonuclear leukocytes and macrophages, which are present in high numbers in chronic wound tissue. Therefore, our findings emphasize the concept that unrestrained protease activity is associated with a nonhealing condition and corroborate the validity of our study. Interestingly, the serine proteinase thrombin was exclusively detected in the healing wound. Thrombin converts fibrinogen monomers into a fibrin network. Besides its essential role in hemostasis, fibrin provides the predominant constituent of the provisional wound matrix and serves as scaffold for cell invasion and granulation tissue formation.1 Therefore, we would argue that in a healing environment mediators of fibrin formation provide markers of a progressing wound healing response. Among the category of protease inhibitors several classes of protease inhibitors were identified in exudates of both healing conditions. In exudate of the normal healing wound, the quantitative value of several proteins belonging to the family of Serine Protease Inhibitors (SERPINs) and Cystatins were increased over those in the exudate obtained from the nonhealing wound. These results emphasize a tissue protective balance of proteases and their inhibitors in the healing condition. The SERPINs have evolved to be the predominant family of serine protease inhibitors in man.30 SERPINs are a superfamily of structurally similar but functionally diverse proteins that regulate a variety of proteolytic events central to tissue repair including phagocytosis, coagulation, fibrinolysis, and complement activation, as well as angiogenesis, apoptosis, and inflammation. In our study, SERPINA1 and SERPINA3 have been identified in high abundance in exudate of the healing wound. Substantial data in the literature indicates that SERPINA1 and SERPINA3 control the activity of proteinases central during tissue repair including neutrophil elastase and cathepsin G, respectively.31,32 Furthermore, decreased activity of both SERPINs has been associated with venous ulcers.27,33 Therefore, we argue that detection of SERPINA1 and -A3 in exudate of the healing wound might be considered a marker of healing. 4764

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Eming et al. In contrast, SERPIND1 was exclusively detected in the exudate of the nonhealing wound. SERPIND1 inhibits thrombin and might therefore delay thrombin-mediated fibrin deposition in the chronic wound environment. This process might contribute to the insufficient formation of a functional provisional wound matrix as outlined earlier. Finally, SERPINF1 was identified with a 3.4-fold increase in exudate of the healing wound versus the nonhealing wound. SEPINF1 has been demonstrated to act as a strong antiangiogenic molecule rather than a protease inhibitor.34 Induction of angiogenesis is a central repair mechanism, in particular during the phase of granulation tissue formation.35 Indeed, impaired angiogenesis and disturbed endothelial cell function is a hallmark of chronic wounds.7,26 The upregulation of several potent pro-angiogenic mediators, including bFGF and VEGFA, has been associated with the phase of tissue formation and are thought to be functionally relevant in wound angiogenesis. On the other hand, a strong pro-angiogenic response in healing wounds has to be tightly counterbalanced by angiogenic inhibitors, in order to avoid an uncontrolled overshooting angiogenic response. However, so far inhibitors of angiogenesis in physiological repair have been less investigated. To our knowledge the presence of SERPINF1 in healing wounds and its potential role as angiogenic regulator during physiological skin repair has not yet been described and merits further investigation. Whether SERPINF1 is a useful biomarker reflecting an efficient repair response in skin remains to be evaluated. A vast number of diverse immune modulatory and stressinduced proteins were identified in exudates of both healing states. In total 23 abundant proteins were identified in this category, of which 7 were exclusively detected in the exudate of the nonhealing wound; 3 were at least 2-fold and another 3 less than 2-fold increased over those in the healing wound. Therefore, there was a clear tendency of increased quantities of immune modulatory and stress-induced mediators in the nonhealing versus the healing wound microenvironment. In this category lactotransferrin was identified as having the highest increase of all proteins identified in the proteome analysis in exudate of the nonhealing wound versus the healing wound. The significant increase of lactotransferrin levels in exudate derived from nonhealing versus healing wounds was confirmed in a larger cohort of exudate samples. The presence of lactotransferrin in nonhealing wounds has not been reported in the literature, and a detailed analysis of its potential function in skin repair has not been performed. Since its purification from milk 50 years ago, lactotransferrin has been assigned diverse functions.36,37 Many of its activities can be grouped under host defense. In particular, its strong antimicrobial activity is thought to be mediated by diverse mechanisms, including its iron scavenging property, its proteolytic activity, and its inhibition of bacterial biofilm formation.37 Furthermore, several studies emphasize its role in regulating cellular signaling pathways, which control inflammation, cytokine expression, and cell cycle arrest. Taken together, although at the present stage it is difficult to speculate on the specific role of lactotransferrin during skin repair, on the basis of our results there is strong evidence that high concentrations in wound exudate are associated with a nonhealing state. Among the immunomodulatory proteins ANXA1 also was identified to be strongly upregulated in the venous leg ulcer. This protein can affect many pathways of the inflammatory response, e.g., leukocytes extravasation and inflammatory cytokine secretion.38 Furthermore, ANXA1 can be induced by

research articles

Comparative Proteome Analysis of Wound Exudates glucocorticoids to target phospholipase A2, inducible nitric oxide synthase, or cyclooxygenase suppressing inflammatory reactions. In addition, ANXA1 promotes anti-inflammatory removal of apoptotic cells by professional phagocytes.39,40 The role of ANXA1 in skin repair is unclear; however, ANXA1 may be needed to resolve inflammation and to stimulate progression into later phases of wound repair. The persistence of increased levels of ANXA1 in chronic wounds potentially reflects the insufficient anti-inflammatory effect of ANXA1, resulting in chronification of the inflammatory response. As a consequence the strong expression of anti-inflammatory acting ANXA1 might be indicative for chronic wounds. Interestingly, among the other annexins we tested (ANXA2, ANXA3, ANXA5), specifically ANXA1 was increased in nonhealing wounds. Interestingly, none of the other annexins we tested (ANXA2, ANXA3, ANXA5) were detected in exudate of nonhealing wounds. Previous experiments in mice showed that these three annexins were expressed in activated macrophages and that ANXA5 was detected at the cell membrane of macrophages at the wound site.41 Macrophages contributing to the inflammatory response in the nonhealing wound may therefore produce but not release ANXA2, ANXA3, and ANXA5 into the wound environment. Alternatively, these annexins may be more susceptible to proteolytic degradation than ANXA1 or are rather bound to the extracellular matrix components of the chronic wound, explaining their absence in the exsudates of nonhealing wounds. Furthermore, several other products of myeloid cells such as leukocyte-derived proteases (neutrophile elastase, proteinase 3, MMP9), myeloperoxidase, and several S100 proteins were all markedly increased in the exudate of the nonhealing versus the healing wound and are suggestive to reflect hallmarks of a chronic inflammatory, tissue destructive microenvironment. In particular, the concentration of the S100A9 protein was shown to be significantly increased in nonhealing versus healing wounds in a larger cohort of exudate samples (P < 0.038). S100 proteins are intracellular Ca2+-binding and Ca2+-modulating proteins that play a role in various Ca2+-mediated cell functions including cell growth and differentiation.42 S100A9 protein has been shown to be upregulated during wound healing in skin,20,43 as well as in other sites of inflammation including psoriasis, lichen ruber, and inflammatory bowel disease.44 Our results emphasize that increased S100A9 protein levels are associated with chronic inflammatory conditions of the skin, which is a hallmark of insufficient tissue repair response. Interestingly, whereas all of the antimicrobial peptides and proteins were increased in the exudate of the nonhealing wound including azurocidin-1,45 bacterial/permeability-increasing protein,46 and Lipocalin,47 solely dermcidin was exclusively detected in exudate of the healing wound.48 The reason for the differential detection of dermcidin in the healing wound is elusive and merits further analysis. The identification of a profound dysbalance toward high proinflammatory and stress-induced mediators versus low growth promoting molecules in the microenvironment of venous ulcers is consistent with the current pathological concept of this disease.23 The current concept suggests that because of the venous insufficiency blood cells are trapped in the venous microcirculation secondary to venous hypertension and release toxic metabolites that lead to complex tissue remodeling and ultimately severe tissue damage. Furthermore, increased hydrostatic pressure was shown to up-regulate and activate a variety of cellular adhesion molecules and their counter recep-

tors, thus enhancing the continuous extravasation of activated leukocytes, specifically neutrophils and macrophages.23 The persistence of these inflammatory cells at the wound site lead to an unrestricted proteolytic activity that is considered the final executor of a pathogenetic chain leading to matrix disruption and proteolysis of growth factors and their receptors. Therefore, modulating and transforming a destructive inflammatory response into a repair-promoting event might offer the development of novel therapeutic strategies for chronic wounds. To date, a comprehensive analysis of pro-inflammatory mediators in chronic ulcers was lacking. Our study broadens the insight on the inflammatory mediators involved in the pathology of repair and contributes to the identification of potential novel therapeutic targets. In future studies, these data will be extended, and the presence of all detected factors will be validated in a larger cohort of wound exudate samples. In addition, their concentrations will be correlated to the clinical outcome of the healing response. Therefore, we propose our analysis as basis for the search for surrogate biomarkers that give rise to a better understanding and prediction of a successful healing response or its failure.

Acknowledgment. We thank Michael Piekarek for technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (S.A.E., T.K., B.B., and M.K.; SFB829), the European Community’s 7th Framework Prgramme, project Angioscaff NMP-LA-2008-214402 (S.A.E.), the Center for Molecular Medicine Cologne (T.K. and M.K.), and University of Virginia Cancer Center (J.W.F.). Supporting Information Available: Link to access mass spectrometric data. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Gurtner, G. C.; Werner, S.; Barrandon, Y.; Lomgaker, M. T. Wound repair and regeneration. Nature 2008, 453, 314–321. (2) Scha¨fer, M.; Werner, S. Transcriptional control of wound repair. Annu. Rev. Cell. Dev. Biol. 2007, 23, 69–92. (3) Werner, S.; Grose, R. Regulation of wound healing by growth factors and cytokines. Physiol. Rev. 2003, 83, 835–870. (4) Fonder, M. A.; Lazarus, G. S.; Cowan, D. A.; Aronson-Cook, B.; Kohli, A. R.; Mamelak, A. J. Treating the chronic wound: A practical approach to the care of nonhealing wounds and wound care dressings. J. Am. Acad. Dermatol. 2008, 58, 185–206. (5) Chen, W. Y.; Rogers, A. A. Recent insights into the causes of chronic leg ulceration in venous diseases and implications on other types of chronic wounds. Wound Repair Regen. 2007, 15 (4), 434–449. (6) Grey, J. E.; Leaper, D.; Harding, K. How to measure success in treating chronic leg ulcers. B.M.J. 2009, 338, b1434. (7) Lauer, G.; Sollberg, S.; Cole, M.; Flamme, I.; Mann, K.; Krieg, T.; Eming, S. A. Expression and Proteolysis of VEGF is increased in chronic wounds. J. Invest. Dermatol. 2000, 115, 12–18. (8) Buchstein, N.; Hoffmann, N.; Smola, H.; Lang, S.; Paulsson, M.; Niemann, C.; Krieg, T.; Eming, S. A. Alternative proteolytic processing of hepatocyte growth factor during wound repair. Am. J. Pathol. 2009, 174 (6), 2116–2128. (9) Moor, A. N.; Vachon, D. J.; Gould, L. J. Proteolytic activity in wound fluids and tissues derived from chronic venous leg ulcers. Wound Repair Regen. 2009, 17 (6), 832–839. (10) Rayment, E. A.; Upton, Z.; Shooter, G. K. Increased matrix metalloproteinase-9 (MMP-9) activity observed in chronic wound fluid is related to the clinical severity of the ulcer. Br. J. Dermatol. 2008, 158 (5), 951–961. (11) Fernandez, M. L.; Broadbent, J. A.; Shooter, G. K.; Malda, J.; Upton, Z. Development of an enhanced proteomic method to detect prognostic and diagnostic markers of healing in chronic wound fluid. Br. J. Dermatol 2008, 158 (2), 281–290. (12) Escalante, T.; Rucavado, A.; Pinto, A. F.; Terra, R. M.; Gutie´rrez, J. M.; Fox, J. W. Wound exudate as a proteomic window to reveal

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