Effects of erythropoietin in skin wound healing ... - The FASEB Journal

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Effects of erythropoietin in skin wound healing are dose related Heiko Sorg,* Christian Krueger,* Torsten Schulz,* Michael D. Menger,† Frank Schmitz,‡ and Brigitte Vollmar*,1 *Institute for Experimental Surgery, University of Rostock, Rostock, Germany; and †Institute for Clinical and Experimental Surgery and ‡Institute of Anatomy, University of Saarland, Homburg-Saar, Germany The hematopoietic growth factor erythropoietin (EPO) attracts attention due to its all-tissueprotective pleiotropic properties. We studied the effect of EPO on dermal regeneration using intravital microscopy in a model of full dermal thickness wounds in the skin-fold chamber of hairless mice. Animals received repetitive low doses or high doses of EPO (RLD-EPO or RHD-EPO) or a single high dose of EPO (SHDEPO). SHD-EPO accelerated wound epithelialization, reduced wound cellularity, and induced maturation of newly formed microvascular networks. In contrast, RHD-EPO impaired the healing process, as indicated by delayed epithelialization, high wound cellularity, and lack of maturation of microvascular networks. Also, RHD-EPO caused an excessive erythrocyte mass and rheological malfunction, further deteriorating vessel and tissue maturation. Moreover, RHD-EPO altered fibroblast and keratinocyte migration in vitro, while both cell types exposed to RLD-EPO, and, in particular, to SHD-EPO showed accelerated wound scratch closure. In summary, our data show that a single application of a high dose of EPO accelerates and improves skin wound healing.—Sorg, H., Krueger, C., Schulz, T., Menger, M. D., Schmitz, F., Vollmar, B. Effects of erythropoietin in skin wound healing are dose related. FASEB J. 23, 3049 –3058 (2009). www.fasebj.org

ABSTRACT

Key Words: microcirculation 䡠 cell migration 䡠 apoptosis 䡠 angiogenesis 䡠 epithelialization 䡠 pericytes Skin wound healing is the physiological process of regenerating dermal and epidermal tissue. Because the skin protects the organism against external influences such as radiation, dehydration, and entry of pathogenic microorganisms (1), early and adequate repair of wounded skin is a basic task of the organism to restitute tissue integrity and homeostasis (2). This comprises a set of events during the temporally and spatially overlapping inflammatory, proliferative, and maturation phases (3). Normal skin wound healing involves specific cell-cell and cell-matrix interactions, which are mediated by growth factors. They enhance tissue repair by improving chemotaxis, cell proliferation, angiogenesis, extracellular matrix deposition, and remodeling. During the final maturation process, the epidermis regenerates 0892-6638/09/0023-3049 © FASEB

from transient hypertrophy, and a dermal collagen matrix replaces the provisional wound matrix (4, 5). This results in an almost acellular and avascular wound (6, 7). The deterioration of the balance between stimulatory and inhibitory events, as known for diabetes, kachexia, and immunosuppression, affects the physiological healing process (4) and results in insufficient repair and hypertrophic scar formation (8, 9). In recent years, several growth factors have been identified to regulate skin wound repair (10). Besides its hematopoietic effects, the growth factor erythropoietin (EPO) has been shown to exert pleiotropic properties, such as cytoprotection, anti-inflammation, and antiapoptosis, in the central nervous and cardiovascular system, as well as in kidney and liver (11–16). Moreover, there is some evidence that EPO amends skin wound healing (17, 18). It has, however, also been reported that EPO enhances abnormal peripheral microvasculature, ischemic tissue damage, and poor wound healing in hemodialysis patients (19). Although a single administration of EPO is not likely to be harmful, the repetitive administration might raise red blood cell count and blood viscosity, thereby affecting hemorheology and local tissue injury. We studied dermal wound healing in mice that received either repetitive low doses (RLD-EPO) or high doses (RHD-EPO) of EPO or a single high dose (SHD-EPO) of EPO only, to determine a safe and effective dosage of EPO in wound regeneration.

MATERIALS AND METHODS Animals A total of 47 male homozygous SKH-1-hr hairless mice (12–16 wk old) with a body weight (bw) of 25–35 g was used for the study. The animals were housed in standard laboratories with a 12 h light-dark cycle and had free ad libitum access to standard laboratory food and water. The experiments were conducted in accordance with the guidelines for the Care and Use of Laboratory Animals and the Institutional Animal Care 1

Correspondence: Institute for Experimental Surgery, University of Rostock, Schillingallee 69a, 18055 Rostock, Germany. E-mail: [email protected] doi: 10.1096/fj.08-109991 3049

and Use Committee (University of Rostock, Medical Faculty, Rostock, Germany). Implantation of the dorsal skin-fold chamber and wounding For intravital microscopy of skin wound healing, the dorsal skin-fold chamber preparation in mice was used (20). Mice were anesthetized by intraperitoneal application of ketamine (90 mg/kg bw; 10% ketamin; Bela-Pharm, Vechta, Germany) and xylazine (25 mg/kg bw; 2% Rompun; Bayer Health Care, Leverkusen, Germany). To implant the chamber, the dorsal skin fold was sandwiched between two symmetric titanium frames. The creation of a full-dermal-thickness wound was achieved after the area was marked with a standardized circular ink stamp (2.5 mm in diameter). The skin was only wounded at one side of the skin fold by removing the complete skin with epidermis and dermis, as well as the subcutis and the panniculus carnosus. The final wound area was ⬃5.0 mm2. After the preparation, the wound was covered with a removable glass coverslip incorporated in one of the titanium frames to prevent desiccation (20). Animals tolerated the chamber well and showed no signs of discomfort or changes in sleeping and feeding habits. Experimental groups and protocol A total of 47 animals with dermal wounds was included in the study; they were randomly allocated into five experimental groups. Animals received a daily intraperitoneal injection of either RLD-EPO [epoetin-␣ of ovarian cells of the Chinese hamster cell line CHO-K1 (Erypo; Janssen-Cilag, Neuss, Germany); 400 U/kg bw; n⫽8] or RHD-EPO (Erypo; 5000 U/kg bw; n⫽12), respectively. Other animals received only an SHD-EPO at the day of wounding (Erypo; 5000 U/kg bw; n⫽7) followed by daily saline applications (0.9% NaCl; 12.5 ml/kg). An additional set of animals was treated with the pan-caspase inhibitor zVAD-fmk (3.3 mg/kg bw; n⫽8; R&D Systems, Wiesbaden, Germany) to evaluate the effect of an almost exclusively antiapoptotic drug on wound healing. Control animals received equivalent volumes of physiological saline (0.9% NaCl; 12.5 ml/kg; n⫽12). Animals were studied by intravital fluorescence microscopy on d 3, 6, 9, and 12 after wounding. At the end of the experiments, blood and wound tissue was collected for subsequent laboratory analysis. Microscopic analysis of wound repair and microcirculation All procedures were performed in ketamine/xylazine-anesthetized mice. Analysis of wound epithelialization was performed under a stereomicroscope using planimetric techniques (IC-A; Leica Microsystems GmbH, Wetzlar, Germany). Wound closure was considered complete when the entire surface area was covered with tissue. Analysis of angiogenesis and microcirculation was performed with the use of an intravital fluorescence epi-illumination microscope (Axiotech Vario; Zeiss, Jena, Germany). Contrast enhancement for microvessel imaging was achieved after retrobulbar injection of 0.1 ml 2% FITC-labeled dextran (molecular mass of 150 kDa; Sigma Chemical, Deisenhofen, Germany; ref. 20) and allowed analysis of microvessel diameter and functional microvessel density (FMD). Analyses were performed offline using a computer-assisted image analysis system (CapImage; Dr. Zeintl Software, Heidelberg, Germany). FMD was defined as the total length of red blood cell-perfused microvessels per observation area (cm/cm2). The 3-dimensional aspect of the wound was considered by focusing through the depth of the wound. 3050

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Histology and immunohistochemistry The titanium frames were explanted on d 12, and the sandwiched skin was fixed in 4% phosphate-buffered formalin for 3 d and then embedded in paraffin. From the paraffin-embedded tissue blocks, 4-␮m sections were serially cut and stained with hematoxylin-eosin for assessment of routine histology and for wound cellularity as a parameter for the resolution of the healing inflammatory tissue (21). For this purpose, digitized images were taken in grayscale format and analyzed by the CapImage software. A random sample of a wound image was taken, and the gray level necessary to mark the complete area of the nuclei of the cells was determined. This gray value served as threshold value for analysis of all further images. For each wound, an identical area of ⬃1.0 mm2 was analyzed. Values for cellularity are given as area (mm2). For the evaluation of apoptotic cell death, we performed both cleaved caspase-3 immunohistochemistry and TUNEL analysis in sections collected on poly-l-lysine-coated glass slides. The sections were treated by microwave for antigen unmasking, followed by overnight exposure at 4°C to rabbit polyclonal anti-cleaved caspase-3 antibody (D175; 1:500; Cell Signaling Technology, Frankfurt, Germany). This antibody detects endogenous levels of the large fragment (17/19 kDa) of activated caspase-3 but not of full-length caspase-3. Then, sections were developed with a horseradish peroxidase-conjugated goat antirabbit IgG (1:20; DakoCytomation, Hamburg, Germany). To determine cell nuclear DNA fragmentation, the TUNEL assay was used according to the manufacturer’s instructions (ApopTag Peroxidase In situ Apoptosis Detection Kit S7101; Chemicon, Hofheim, Germany). 3,3⬘-Diaminobenzidine (S 3000; DakoCytomation) was used as chromogen. Leukocytes were stained with the AS-D chloroacetate esterase (CAE) technique and were identified by positive staining and morphology within the healing tissue. For CD31-staining, goat polyclonal anti-CD31 (1:50; Santa Cruz Biotechnology, Heidelberg, Germany) was used as primary antibody at 4°C overnight, followed by exposure to a secondary antibody at room temperature (LSAB Kit; DakoCytomation), according to the manufacturer’s instructions. New fuchsin (DakoCytomation) was used as chromogen. All sections were counterstained with hemalaun and examined by light microscopy (Axioskop 40; Zeiss). The numbers of cleaved caspase-3-, TUNEL-, and CAE-positive cells were counted in 5 to 6 directly neighboring high power fields (HPFs) within the wound granulation tissue and are given as n/HPF. The wound granulation tissue as well as the initial wound margins could be exactly differentiated by a clear demarcation of the granulation tissue against the noninjured skin (20). Microvessel density (n/HPF) was assessed by counting vascular lumina with CD31-positive endothelial lining within the wound granulation tissue. Transmission electron microscopy In parallel to the intravital microscopic experiments, additional experiments (n⫽4 animals/group) were performed, and skin wounds were processed for transmission electron microscopic analysis. Specimens were fixed with 2% glutaraldehyde buffered with 0.10 M sodium cacodylate (pH 7.4). The postfixation was performed in 3 steps using 1% osmium tetroxide in 100 mM sodium cacodylate (pH 7.4) for 1 h at 4°C. After dehydration in an ascending concentration series of ethanol and infiltration with propylene oxide, specimens were finally embedded in Araldite 502 (Serva, Heidelberg, Germany). Semithin (for light microscopic control) or ultrathin sections were prepared on an ultramicrotome (Ultracut;

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SORG ET AL.

SWS, Leica). The ultrathin sections were stained with 2% uranyl acetate in H2O for 15 min and examined in a Tecnai G2 transmission electron microscope (FEI, Hillsboro, OR, USA) operated at 100 kV. In vitro cell migration assay To evaluate the effect of EPO on cell migration, fibroblasts (L929) and keratinocytes (HaCaT) were seeded onto Petri dishes (5⫻105 cells/dish) and grown to confluence in lowglucose DMEM (PAA, Co¨lbe, Germany), 10% FCS, and 1% penicillin/streptomycin. After the removal of the medium, cell monolayers were scratched with a pipette tip (10 ␮l). Then, the cells were reexposed to medium supplemented with either 10% FCS or EPO at two different concentrations: RLD-EPO, with 5 U/ml, and RHD-EPO, with 50 U/ml. To simulate the application of a single high dose of EPO, cells were exposed to medium supplemented with 50 U/ml EPO for the first 24 h, followed by removal of the EPO-containing medium and reexposure to EPO-free medium with 10% FCS (SHD-EPO). EPO doses were calculated according to the EPO doses given in vivo. In vivo, low-dose EPO was 400 U/kg, which represents ⬃10 U/mouse. Given a circulating blood volume of 7– 8% bw (⬃2 ml), this represents 5 U/ml EPO. Accordingly, this dose was given in the in vitro setting as the low dose. The high-dose EPO in vivo was ⬃10⫻ of the low dose. Accordingly, we applied 50 U/ml EPO in the high-dose in vitro setting. As a positive stimulant for fibroblast and keratinocyte migration, we used basic fibroblast growth factor (bFGF; 20 ng/ml; R&D Systems) and keratinocyte growth factor (KGF; 10 ng/ml; Sigma), respectively. The scratch area was photographed immediately and at 24, 48, and 72 h after scratching. The digitized images were taken in grayscale format and analyzed densitometrically by the CapImage software. A random sample of a scratch area was taken, and the gray level necessary to mark the complete area of the cells migrated into the scratch area was determined. This gray value served as the threshold value for analysis of all further images. Values of cell migration into the scratch area are given as the area covered by cells in the percentage of the initial scratch area. All experiments were performed in triplicate. Laboratory analysis At the end of the experiment, blood was sampled for analysis of systemic red and white blood cell counts, hemoglobin concentration, and hematocrit using an automatic cell counter. Statistical analysis All data are given as means ⫾ se. Data were analyzed for normality and equal variance across groups. After the multivariate analysis of interaction (2-way ANOVA) between time and groups, where appropriate, differences between groups at a single point of time were tested separately by using 1-way ANOVA or ANOVA on ranks followed by the appropriate post hoc comparison test including Bonferroni probabilities to compensate for multiple comparisons. To test for time effects within each group, the repeated measures ANOVA approach was applied followed by the appropriate post hoc comparison test including correction of the ␣ error according to the Bonferroni probabilities for repeated measurements. Overall statistical significance was set at P ⬍ 0.05. For clarity and rapid interpretation of data, only significant differences for comparison between groups at a single point of time are given. Statistics were performed using the software package SigmaStat 10.0 (Jandel Corporation, SanRafael, CA, USA). EPO IN SKIN WOUND HEALING

RESULTS Wound epithelialization The initial size of the wound, with an average area of 5.0 ⫾ 0.3 mm2, was comparable in all groups. At the subsequent observations, planimetric analysis of the wound area in the control group showed a continuous epithelialization, with 50 ⫾ 5% wound coverage at d 6 and complete wound closure (100⫾0%) at d 12 (Fig. 1). SHD-EPO significantly accelerated wound epithelialization, with 73 ⫾ 9% wound coverage at d 6 and almost complete wound closure (92⫾3%) at d 9 (Fig. 1). In contrast, RHD-EPO significantly delayed epithelialization, with only 32 ⫾ 3 and 64 ⫾ 7% wound closure at d 6 and 9, and did not allow complete wound closure at d 12 (Fig. 1). Epithelialization after RLD-EPO and zVAD was similar to that observed in controls (Fig. 1). Microvascular features Intravital microscopic analyses revealed newly formed microvessels arranged in a circular and radial pattern (Fig. 2). In control animals, the diameters of circular vessels decreased over the entire 12-d observation period, indicating vessel maturation (Fig. 2E). Circular vessels of RLD-EPO- and SHD-EPO-treated animals initially showed increased diameters but then also demonstrated a diameter reduction as seen in controls (Fig. 2E). In contrast, RHD-EPO increased the diameters of the circular vessels until the end of the observation period (Fig. 2E). The radial vessels were smaller in diameter than the circular ones (Fig. 2F). Interestingly, at d 3, radial vessels could be observed in the wounds of only 25% of controls and in none of the wounds of the animals treated with either EPO or zVAD (Fig. 2F). At d 6, however, radial vessels were found developed in all wounds and did not significantly differ in diameter among groups until d 9 (Fig. 2F). Of interest, only animals with repetitive EPO application exhibited a significant increase of vessel diameters at d 12. In controls, but also in RLD-EPO- and SHD-EPOtreated animals, the functional density of circular vessels decreased to ⬃50 cm/cm2, while in zVAD- and RHD-EPO-treated animals, the density of circular vessels remained ⬎140 cm/cm2 (Fig. 2G). The density of radial vessels did not significantly differ among the groups over time (Fig. 2H). Wound tissue histology and immunohistochemistry Histological analysis revealed significantly lower cellularity in the wounds of RLD-EPO- and SHD-EPO-treated animals when compared with RHD-EPO- and zVAD-treated animals. This supports the hypothesis of a maturation process in RLD-EPO- and SHD-EPO-treated wound tissue, with a shortened inflammatory phase and an accelerated resolution of the early granulation tissue (Fig. 3A, B). In line with this interpretation, the granulation tissue of RHD-EPO-treated animals still presented with a markedly increased leukocytic tissue infiltration on d 12, which was 3051

2- to 3-fold higher than that in controls and SHD-EPOtreated animals (Fig. 3C, D). The evaluation of cell apoptosis at d 12 showed slightly reduced numbers of apoptotic cells in wounds of all EPO-treated animals compared with controls. Although not statistically significant, wounds of zVADtreated animals revealed the most pronounced reduction of cellular apoptosis (Fig. 4). With the use of immunostaining of the endothelial cell marker CD31 as a parameter of angiogenesis, microvessel density within the wound granulation tissue of RHD-EPO-treated animals averaged 40 ⫾ 6 microvessels/HPF, significantly higher than that of the other groups (⬃17 microvessels/HPF; Fig. 5). This further confirmed that RHD-EPO delays maturation within the newly formed microvasculature. Transmission electron microscopy of wound tissue Endothelial cells and pericytes were regularly observed in almost all microvessels in the control, RLD-EPO, and SHD-EPO groups. Pericytes closely covered the microvessels, and the basal lamina presented as a compact dense layer in these wounds (Fig. 6A, B). In contrast, microvessels in wounds of RHD-EPO-treated animals frequently impressed with a sole endothelial lining and absence of pericytes. If pericytes were present, they occasionally showed only loose contact with the endothelial cells. In addition, basal lamina were mostly lacking in these microvessels, underlining the incomplete maturation process after RHD-EPO treatment (Fig. 6C, D). In vitro cell migration assay

Figure 1. Representative photomacroscopic images (A) and quantitative planimetric analysis (B) of wounds during healing in mice treated with either saline daily (control; 0.9% NaCl; 12.5 ml/kg bw; n⫽12), RLD-EPO daily (400 U/kg bw; n⫽8), RHD-EPO daily (5000 U/kg bw; n⫽12), or SHD-EPO given on day of wounding (5000 U/kg bw; n⫽7). Additional animals (n⫽8) received the pan-caspase inhibitor zVAD-fmk daily (zVAD; 3.3 mg/kg bw). A) Note the continuous process of wound closure, with complete epithelialization over d 6 (control d6) to d 12 (control d12) in a saline-treated animal and the significantly accelerated wound closure in an SHDEPO-treated wound (SHD-EPO d6), which has already completed at d 9 (SHD-EPO d9). In contrast, the wound in the RHD-EPO-treated animal shows delayed epithelialization (RHD-EPO d6) and only incomplete coverage at d 12 (RHDEPO d12). Dotted lines indicate edges of wounds at day of wounding; dashed line indicates open wound area at d 12 in RHD-EPO-treated animal. Scale bars ⫽ 1000 ␮m. B) Planimetric analysis confirms accelerated wound healing in SHDEPO-treated animals and delayed healing after RHD-EPO treatment. Values are means ⫾ se; 1-way ANOVA and HolmSidak test. *P ⬍ 0.05 vs. control; #P ⬍ 0.05 vs. RHD-EPO; §P ⬍ 0.05 vs. RLD-EPO; $P ⬍ 0.05 vs. SHD-EPO.

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Migration of fibroblasts and keratinocytes was significantly increased by bFGF and KGF, respectively. SHDEPO also significantly increased both fibroblast and keratinocyte migration compared with 10% FCS, while RLD-EPO increased only fibroblast migration significantly (Fig. 7). In contrast, RHD-EPO significantly impaired in vitro migration of both cell types (Fig. 7), confirming the results of the in vivo experiments. Laboratory analysis Blood samples of saline-, zVAD-fmk-, and SHD-EPOtreated animals revealed physiological values of erythrocyte count, hemoglobin concentration, and hematocrit (Table 1). In RLD-EPO- and RHD-EPO-treated animals, the erythrocyte count increased significantly in a dosedependent manner (Table 1). In all EPO-treated groups, the white blood cell count was also elevated significantly when compared with controls and zVAD-treated animals (Table 1).

DISCUSSION The present in vivo study on skin wound healing in hairless mice shows that SHD-EPO causes acceleration of wound epithelialization; shortening of the inflammatory phase, with faster resolution of the


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Figure 2. A–C) Representative intravital fluorescence microscopic images of microcirculation at d 6 of wound healing in an RHD-EPO-treated animal. D) Microcirculation FMD of nonwounded skin. E–H) Quantitative analysis of diameter (␮m) and FMD (cm/cm2) in circular (E, G) and radial vessels (F, H). Animals were treated with either saline daily (control; 0.9% NaCl; 12.5 ml/kg bw; n⫽12), RLD-EPO daily (400 U/kg bw; n⫽8), RHD-EPO daily (5000 U/kg bw; n⫽12), or SHD-EPO on day of wounding (5000 U/kg bw; n⫽7). Additional animals (n⫽8) received the pan-caspase inhibitor zVAD daily (3.3 mg/kg bw). The process of wound healing shows distinct patterns of newly formed microvascular networks (A). A first microvascular network creates an inner circular ring of vessels at the wound margin (A; higher magnification in B). This is surrounded by outer radially localized vessels, supplying the vasculature within the inner circular ring (A; higher magnification in C). Nonwounded skin (D) presents with the typical cutaneous microvascular architecture, i.e., capillaries forming dermal papillary loops around empty hair follicles (arrowheads). Continuous line (A) marks edge of the wound at day of wounding; dashed line indicates border between radial and circular microvessels. Scale bars ⫽ 200 ␮m (A); 80 ␮m (B–D). Values are means ⫾ se; 1 -way ANOVA and Holm-Sidak test. *P ⬍ 0.05 vs. control; #P ⬍ 0.05 vs. RHD-EPO; §P ⬍ 0.05 vs. RLD-EPO; $P ⬍ 0.05 vs. SHD-EPO.

early granulation tissue; and decreased vessel density with vessel maturation at the wound site. In contrast, RHD-EPO exerts harmful effects, as shown by the overall delay in wound healing. With the use of in vitro assays, the study further shows that migration of keratinocytes and fibroblasts is stimulated by RLDEPO, and especially SHD-EPO, whereas RHD-EPO EPO IN SKIN WOUND HEALING

does not improve cell migration. Because the antiapoptotic treatment with zVAD showed, at least in part, comparable effects on wound healing as observed after RHD-EPO, the antiapoptotic environment might account for the delay in tissue and vessel maturation during wound healing. Furthermore, the delay in wound healing after RHD-EPO may be 3053

Figure 3. Representative images (A, C) and quantitative analysis (B, D) of hematoxylin-eosin staining for cellularity (A, B) and leukocyte infiltration (CAE; C, D) in wound tissue specimens at d 12 after wounding. Animals were treated with either saline daily (control; 0.9% NaCl; 12.5 ml/kg bw; n⫽12), RLD-EPO daily (400 U/kg bw; n⫽8), RHD-EPO daily (5000 U/kg bw; n⫽12), or SHD-EPO on day of wounding (5000 U/kg bw; n⫽7). Additional animals (n⫽8) received the pan-caspase inhibitor zVAD daily (3.3 mg/kg bw). Scale bars ⫽ 20 ␮m (A); 50 ␮m (C). Values are means ⫾ se; 1-way ANOVA and Holm-Sidak test. *P ⬍ 0.001 vs. control; #P ⬍ 0.001 vs. RHD-EPO; §P ⬍ 0.001 vs. RLD-EPO.

attributed to the increase of erythrocyte mass, which is associated with undesirable hemorheological effects. Previous studies (22–24) have indicated that EPOinduced excessive erythrocytosis aggravates acute ischemic injury and leads to hepatic, renal, neuronal, and muscular degeneration. In contrast, low-dose EPO

Figure 4. Quantitative analysis of cleaved caspase-3 and TUNEL for evaluation of apoptotic cell death in wound tissue specimens at d 12 after wounding. Animals were treated with either saline daily (control; 0.9% NaCl; 12.5 ml/kg bw; n⫽12), RLD-EPO daily (400 U/kg bw; n⫽8), RHD-EPO daily (5000 U/kg bw; n⫽12), or SHD-EPO on day of wounding (5000 U/kg bw; n⫽7). Additional animals (n⫽8) received the pan-caspase inhibitor zVAD (3.3 mg/kg bw). Values are means ⫾ se; 1-way ANOVA and Holm-Sidak test. 3054

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treatment, which is not associated with erythrocytosis, has been shown capable of protecting from postischemic injury (25–28). Accordingly, the improvement of wound healing by RLD-EPO and SHD-EPO treatment, as observed in the present study, most probably can be attributed to the absence of excessive erythrocytosis. In contrast, the pronounced erythrocytosis observed in the RHD-EPO-treated animals may be the cause for the deteriorated healing process. In line with this view, high values of hematocrit are known to adversely affect rheological properties, to impair nutritive perfusion and metabolic functions of injured tissue (29, 30), and to promote the development of endothelial damage (31). Moreover, the elevation of the hematocrit by EPO is reported to be associated with increased blood pressure (32) and increased thrombosis rate (33, 34), both representing conditions that contribute to impaired regeneration and remodeling (35). Previous studies (36 –38) have demonstrated that multiple doses of EPO had no added therapeutic effect when compared with a single dose or repeated low-dose administration. In our study, RLD-EPO, with a cumulative dose of ⬃5000 U/kg, and, in particular, SHD-EPO, with a single application of 5000 U/kg, improved skin wound healing, while RHD-EPO, with a cumulative dose of ⬃60,000 U/kg, was found to deteriorate the dermal healing process. Thus, our study also confirms that a low dose of EPO, which can be applied by single injection, can exert pleiotropic effects without concomitant erythrocytosis. Although angiogenesis and a degree of inflammation are required for significant repair, their resolution is


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Figure 5. Representative images (A) and quantitative analysis (B) of CD31-stained endothelial lining (A, arrowheads) to determine microvessel density in wound tissue specimens at d 12 after wounding. Animals were treated with either saline daily (control; 0.9% NaCl; 12.5 ml/kg bw; n⫽12), RLD-EPO daily (400 U/kg bw; n⫽8), RHD-EPO daily (5000 U/kg bw; n⫽12), or SHD-EPO on day of wounding (5000 U/kg bw; n⫽7). Additional animals (n⫽8) received the pan-caspase inhibitor zVAD daily (3.3 mg/kg bw). Scale bars ⫽ 100 ␮m. Values are means ⫾ se; 1-way ANOVA and Holm-Sidak test. *P ⬍ 0.001 vs. control; #P ⬍ 0.001 vs. RHD-EPO; §P ⬍ 0.001 vs. RLD-EPO.

also required (21). This is in line with studies (21) demonstrating that increased cellularity is associated with decreased biomechanical strength. Key elements of wound remodeling are dense collagen packing,

decline in vascular density, and loss of cells via emigration, apoptosis, and necrosis (4, 39). Thus, the reduced cellularity, leukocyte infiltration, and vessel density as well as the pericyte coverage of microvessels after SHD-EPO treatment indicate a faster and adequate maturation and remodeling compared with RHD-EPO. In fact, RHD-EPO-associated high tissue leukocyte infiltration might account for the impaired healing, as has been demonstrated for prolonged and excessive inflammatory reactions (40, 41). By monitoring CD31 expression (42), tissue transglutaminase (43), and VEGF wound content (44), other studies have described the proangiogenic effect of EPO. In line with these reports, wounds of RHD-EPOtreated animals also presented with higher functional microvessel density and CD31 expression. Enhancement of neovascularization by EPO has been attributed to a direct stimulatory activity of EPO on VEGF and on endothelial cell mitosis (44). EPO was further found to stimulate the proliferation and tube formation of cultured neonatal microvascular endothelial cells (45). In addition, EPO has been shown to enhance inflammation- and ischemia-induced neovascularization, most probably due to a significant increase of the numbers of endothelial progenitor cells in bone marrow, spleen, and peripheral blood (46). Further, EPO enhances the proliferation and migration and reduces the apoptosis of mature endothelial cells (47) and adult endothelial progenitor cells (48). The interesting aspect of EPO-mediated angiogenesis, however, is the fact that the networks in RHD-EPOtreated wounds neither mature nor regress within the time frame of these experiments. Wounds of RLD-EPOand SHD-EPO-treated animals, as well as of control animals, showed a progressive decline in density and diameter of circular vessels as a characteristic feature of wound maturation (4, 20). In contrast, RHD-EPOtreated wounds presented with a constant increase of circular vessel density and a failure in diameter reduction, which has to be interpreted as a delay in vessel

Figure 6. Representative transmission electron microscopic images of newly formed microvessels within wound granulation tissue at d 12 after wounding of an animal treated daily with RLD-EPO (400 U/kg bw; A, B) and of an animal treated with RHD-EPO (5000 U/kg bw; C, D). Note basement membrane (arrows) and pericyte coverage of microvessel in panel B, while in panel D, pericytes are only in loose contact (asterisks) with endothelial cells. In the RLDEPO-treated animal (A, B), pericytes are covered with basement membrane on both their inner (luminal; arrows) and outer (abluminal; arrowheads) sides. E, endothelial cell; P, pericyte; er, erythrocyte; L, lumen. Scale bars ⫽ 5 ␮m (A, B); 2 ␮m (C, D).

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Figure 7. Quantitative assessment of in vitro cell migration using the wound scratch assay with fibroblasts (L929) (A) and keratinocytes (HaCaT) (B) over an observation period of 72 h. Cells were grown to confluence in 10% FCS on Petri dishes and then scratched with a pipette tip (C). Cells were treated with either 10% FCS, 20 ng/ml bFGF (fibroblasts only), 10 ng/ml KGF (keratinocytes only), 5 U/ml EPO (RLD-EPO), 50 U/ml EPO (RHD-EPO), or an initial supplementation with 50 U/ml EPO for the first 24 h with removal of the EPO-containing medium and reexposure to EPO-free medium with 10% FCS (SHD-EPO). Cells were photographed immediately and at 24, 48, and 72 h after the scratch, as representatively shown for fibroblasts among groups at 72 h after scratching (C). Scale bars ⫽ 400 ␮m. Values are means ⫾ se; 1-way ANOVA and Holm-Sidak test. *P ⬍ 0.05 vs. control; #P ⬍ 0.05 vs. RHD-EPO; § P ⬍ 0.05 vs. RLD-EPO; $P ⬍ 0.05 vs. bFGF or KGF.

maturation (49, 50). The initial increase of vessel diameters in all groups except the control group might reflect the proproliferative and proangiogenic effects of EPO (51–53). In fact, RLD-EPO and SHD-EPO may have stimulated initial regeneration, resulting in a transient VEGF-mediated increase of vessel diameters. After RHD-EPO, the significant increase of the hematocrit with subsequent elevation of blood cell viscosity and worsening of rheology may sustain the increase of vessel diameters, with subsequent failure of maturation (29, 32–35). However, it is also possible that the diameter enlargement is caused by impaired diameter control, due to an initial eNOS up-regulation in RLD-EPO and SHD-EPO treatment at the early observation time

points, and continuous eNOS overexpression in RHDEPO treatment throughout the entire observation period (54). For the process of vessel maturation, the coverage of newly formed vessels with mural cells such as pericytes is crucial (55). After the formation of new vessels, the maturation process proceeds with the recruitment of mesenchymal cells differentiating into mature pericytes around the vessel wall (49). Blood vessels, which have no pericyte coverage, are reported to vary extensively in diameter (56) and might finally regress (57). In line with this, electron microscopic analysis revealed some ultrastructural pecularities of microvessels in wound granulation tissue of the RHD-EPO-treated animals,

TABLE 1. Systemic blood cell counts in mice at d 12 after wounding treated with either saline daily, RLD-EPO daily, RHD-EPO daily, or SHD-EPO on day of wounding Treatment

Red blood cells (⫻1012/L)

Hemoglobin (g/dl)

Hematocrit (%)

White blood cells (⫻109/L)

Control RLD-EPO RHD-EPO SHD-EPO zVAD

8.2 ⫾ 0.2 10.6 ⫾ 0.2* 13.0 ⫾ 0.2*,§ 8.6 ⫾ 0.5#,§ 7.9 ⫾ 0.2#,§

13.0 ⫾ 0.3 17.7 ⫾ 0.5* 21.7 ⫾ 0.3*,§ 14.0 ⫾ 0.8#,§ 12.4 ⫾ 0.3#,§

44 ⫾ 1 63 ⫾ 2* 82 ⫾ 1*,§ 48 ⫾ 3#,§ 41 ⫾ 1#,§

4.1 ⫾ 0.4 6.5 ⫾ 0.4* 6.7 ⫾ 0.7* 6.7 ⫾ 0.4* 4.3 ⫾ 0.5#,§,$

Control: 0.9% NaCl; 12.5 ml/kg bw; n ⫽ 12. RLD-EPO: 400 U/kg bw daily; n ⫽ 8. RHD-EPO: 5000 U/kg bw daily; n ⫽ 12. SHD-EPO: 5000 U/kg bw; n ⫽ 7. Additional animals (n⫽8) received the pan-caspase inhibitor zVAD-fmk daily (3.3 mg/kg bw). Values are means ⫾ se; 1-way ANOVA and Holm-Sidak test. *P ⬍ 0.05 vs. control; #P ⬍ 0.05 vs. RHD-EPO; §P ⬍ 0.05 vs. RLD-EPO; $P ⬍ 0.05 vs. SHD-EPO.

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such as loose contact between pericytes and endothelial cells as well as less dense capillary basement membrane, hinting at delayed vessel maturation. Skin wound closure by epithelialization is a major component of wound healing, which depends on the ability of keratinocytes to free themselves, migrate, and readhere to neighboring cells and the basement membrane (58). Therefore, we studied fibroblasts and keratinocytes in vitro and observed enhanced migration with SHD-EPO, while RHD-EPO reduced the migration activity. It has been shown that EPO inhibits the TGF␤1-mediated decrease of intercellular contacts (zona occludens, cadherin) in renal fibrosis (59). In addition, EPO diminishes the expression of Smad-2 (59), which represents a crucial signaling mediator in regulating keratinocyte migration and epithelialization (60). Thus, it seems reasonable that the delay in wound healing with RHD-EPO might be due to Smad-2 down-regulation; however, further exploration is needed. In conclusion, our data show that repetitive low doses but especially single high doses of EPO improve the process of wound healing. Repetitive application of high doses of EPO, in contrast, deteriorate wound healing, most probably by erythrocytosis-associated alteration of tissue and vessel maturation. We thank Berit Blendow, Doris Butzlaff, Dorothea Frenz, and Maren Nerowski (Institute for Experimental Surgery, University of Rostock) for excellent technical assistance; Karin Gerber and Kathrin Sievert-Ku¨chenmeister (Institute for Experimental Surgery, University of Rostock) for help with the animal care; Christina Marx (Institute for Clinical and Experimental Surgery, University of Saarland) for assistance in sample preparation for transmission electron microscopy; Gerlinde Ku¨hnreich (Institute for Anatomy, University of Saarland) for making ultrathin sections; and Marika Fleischer (Language Center, University of Rostock) for critical editing of the manuscript. This work was supported by a grant from the Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg (Vo 450/10-1). We declare no conflicts of interest.

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Received for publication March 25, 2008. Accepted for publication April 9, 2009.

SORG ET AL.