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Original A rticle Transplantation of Endothelial Progenitor Cells Accelerates Dermal Wound Healing with Increased Recruitment of Monocytes/Macrophages and Neovascularization Wonhee Suh, Koung Li Kim, Jeong-Min Kim, In-Soon Shin, Young-Sam Lee, Jae-Young Lee, Hyung-Suk Jang, Jung-Sun Lee, Jonghoe Byun, Jin-Ho Choi, Eun-Seok Jeon, Duk-Kyung Kim Department of Medicine, Samsung Medical Center, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Seoul, Korea Key Words. Endothelial progenitor cell • Macrophage • Monocyte • Neovascularization • Wound healing
Abstract Endothelial progenitor cells (EPCs) act as endothelial precursors that promote new blood vessel formation and increase angiogenesis by secreting growth factors and cytokines in ischemic tissues. These facts prompt the hypothesis that EPC transplantation should accelerate the wound-repair process by facilitating neovascularization and the production of various molecules related to wound healing. In a murine dermal excisional wound model, EPC transplantation accelerated wound re-epithelialization compared with the transplantation of mature endothelial cells (ECs) in control mice. When the wounds were analyzed immunohistochemically, the EPCtransplanted group exhibited significantly more monocytes/ macrophages in the wound at day 5 after injury than did the EC-transplanted group. This observation is consistent with
enzyme-linked immunosorbent assay results showing that EPCs produced in abundance several chemoattractants of monocytes and macrophages that are known to play a pivotal role in the early phase of wound healing. At day 14 after injury, the EPC-transplanted group showed a statistically significant increase in vascular density in the granulation tissue relative to that of the EC-transplanted group. Fluorescence microscopy revealed that EPCs preferentially moved into the wound and were directly incorporated into newly formed capillaries in the granulation tissue. These results suggest that EPC transplantation will be useful in dermal wound repair and skin regeneration, because EPCs both promote the recruitment of monocytes/macrophages into the wound and increase neovascularization. Stem Cells 2005;23:1571–1578
Introduction
ing, monocytes/macrophages play pivotal roles by phagocytosing debris and secreting a large number of cytokines and growth factors, thereby regulating fibroblast migration, proliferation, and subsequent collagen synthesis. Their functional importance has been demonstrated in monocyte/macrophage-depleted animals, which exhibit defective wound repair, such as delays in angiogenesis and re-epithelialization [3]. In the next angiogenesis phase, new blood vessels form in response to an increase in the produc-
Cutaneous wound healing is a complex process involving the interplay of different cell types in the wounded tissues, including inflammatory cells, fibroblasts, keratinocytes, and endothelial cells [1, 2]. All these cells mediate their functions by releasing a variety of chemo-cytokines and growth factors in a cell type– specific manner to initiate inflammation, new blood vessel formation, and tissue remodeling. In the early stage of wound heal-
Correspondence: Duk-Kyung Kim, M.D., Ph.D., Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Ilwon-dong, Kangnam-ku, Seoul 135-710, Korea. Telephone: 82-2-3410-3419; Fax: 82-2-3410-3849; e-mail: dkkim@smc. samsung.co.kr Received December 1, 2004; accepted for publication May 24, 2005; first published online in Stem Cells EXPRESS August 4, 2005. © AlphaMed Press 1066-5099/2005/$12.00/0 doi: 10.1634/stemcells.2004-0340
Stem Cells 2005;23:1571–1578 www.StemCells.com
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tion of angiogenic growth factors and various cytokines by macrophages and keratinocytes. Newly formed vessels not only allow leukocyte migration into the wound, but also supply the oxygen and nutrients necessary to sustain the growth of granulation tissues. In the final tissue remodeling phase, wound contraction and extracellular matrix reorganization occur over several months, converting granulation tissues into a mature scar. Overall, efficient wound healing involves numerous factors, especially a sufficient supply of growth factors and adequate circulation of oxygenated blood. Endothelial progenitor cell (EPC)–assisted regeneration and repair of ischemic tissues have been illustrated in many reports, wherein circulating EPCs are incorporated into the injured vasculature, promoting neovascularization and subsequent functional recovery of the surrounding tissues [4, 5]. Interestingly, recent studies demonstrated the existence of two different EPC subpopulations that are distinct from each other in terms of cell growth potential and origin of cell lineage. In detail, late EPCs with high proliferative capacity and typical endothelial characteristics are derived from hematopoietic stem cells containing a CD34- or CD133-positive cell population, whereas early EPCs with low growth potential are derived from the ex vivo culture of a CD34-negative mononuclear cell population [6, 7]. In particular, the culture-committed early EPCs directly incorporate into neovasculature and also augment angiogenesis through the secretion of angiogenic growth factors, such as vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), monocyte chemotactic protein (MCP)–1, and macrophage inflammatory protein (MIP)–1α [6, 8, 9]. These properties of early EPCs prompted us to hypothesize that transplantation of early EPCs should be useful in wound repair by secreting various wound healing–related cytokines, as well as by enhancing neovascularization. Whereas recent studies have illustrated that bone marrow–derived stem cells participate in cutaneous wound healing and skin regeneration, they have shown little therapeutic advantages in excisional wound models, especially made with normal mice [10, 11]. These results might demonstrate that the woundhealing process is not only affected by stem cells recruited from outside sources, but also regulated by local resident cells in the skin. In this regard, immunodeficient nude mice were engrafted with human peripheral blood–derived early EPCs that should stimulate local resident cells in the skin by supplying a variety of chemo-cytokines. Although human peripheral blood–derived early EPCs have been described to restore the organ vascularization in other tissue ischemia models, the effective contribution of such cells to cutaneous wound repair has yet to be clarified in a preclinical animal model. Herein, we estimated the dermal wound healing effect of human early EPCs and characterized the EPC-assisted wound-healing process by investigating the chemocytokine secretion and the extent of neovascularization.
EPC Transplantation for Dermal Wound Healing
Materials and Methods Cell Culture Peripheral blood mononuclear cells from human volunteers were isolated by density gradient centrifugation and cultured on gelatin-coated plates in endothelial cell basal medium 2 (EBM-2) supplemented with endothelial growth medium (EGM) 2 MV, SingleQuot (Clonetics, Cambrex, Walkersville, MD, http://www. cambrex.com) [12]. After 7 days of culture, EPCs were harvested for the following experiments. The EPC phenotype was confirmed by staining with 1,1'-dilinoleyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)–labeled acetylated low-density lipoprotein (DiI-acLDL; Molecular Probes, Inc., Eugene, OR, http://probes.invitrogen.com) and fluorescein isothiocyanate (FITC)–conjugated Ulex europaeus agglutinin (FITC-ulexlectin; Sigma, St. Louis, http://www.sigmaaldrich.com). Dualstained cells positive for both DiI-acLDL and FITC-ulex-lectin were identified as EPCs, as previously published [9]. Human dermal microvascular endothelial cells (HDMECs) were purchased from Clonetics and cultured in EGM until passages 4–7. Dermal fibroblast cells (HDF-A; Modern Tissue Technology, Seoul, Korea, http://www.biomtt.com) were cultured in fibroblast growth medium 2 (Clonetics). For in vivo wound-healing experiments, HDMECs and EPCs (after 7 days of culture) were harvested and resuspended at 1 × 105 cells in 50 μl of phosphate-buffered saline (PBS). To detect EPC incorporation, HDMECs and EPCs were prelabeled with fluorescent DiI (Molecular Probes, Inc.) by incubating them with 2.5 mg/L DiI in PBS for 10 minutes at 37°C. DiI-labeled cells were resuspended in an identical manner.
Animals and Wound Model All procedures were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH publication no. 8523, revised 1996). To investigate the effect of EPCs on wound healing, male athymic nude mice (Charles River Laboratories, Yokohama, Japan, http://www.criver.com), aged 6–8 weeks and weighing 17–22 g, were anesthetized with an intraperitoneal injection of 100 μl of solution containing 2.215 mg of ketamine and 0.175 mg of xylazine. After the dorsal surface of each mouse had been disinfected with an alcohol swab, a fullthickness excisional wound (0.5 cm in diameter) was created on the dorso-medial back of each animal, using a standard skin biopsy punch (Acuderm Inc., Fort Lauderdale, FL, http://www. acuderm.com) and left undressed. Immediately after surgery, EPCs, HDMECs (1 × 10 5 cells in 50 μl of PBS), or PBS was injected into three different sites of intact dermis nearby the created wound (the total volume of three injections per wound was 50 μl). Injections were placed about 1 cm away from the wound edge to avoid any leakage.
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Analysis of Wound-Closure Rate Wound size was documented with a digital camera (Sony Cybershot F-505V; Carl Zeiss, Tokyo, http://www.zeiss.com) on days 0, 3, 5, 7, 9, and 12. Images were analyzed using Image-Pro Plus (Media Cybernetics, Inc., Silver Spring, MD, http://www.mediacy.com) by tracing the wound margin with a fine-resolution computer mouse and calculating the pixel area. Pixel counts were then related to a circular filter paper of the same diameter as the original wound, which served as a reference for every image. The measurement was performed in duplicate, and the wound-closure rate was expressed as a ratio of the wounded area at each time point divided by the area of the original wound at time 0.
Tissue Preparation Wounds with a 1- to 2-mm margin of normal skin surrounding them were excised and longitudinally cut in half through the leasthealed portion. Both halves of the wound were fixed in 4% paraformaldehyde (Sigma), embedded in paraffin, and serially sectioned (5-μm thickness) perpendicular to the wound surface. Sections with the largest epithelial gap (maximal distance between the epithelial edges) were considered as central sections of the wounds. Every 20th section (100-μm interval) around the central wound was then subjected to the quantification of wound volume and immunohistochemistry. The number of sections examined for following experiments ranges from 10 to 12 per wound.
Analysis of Wound Volume For the measurement of wound volume, sections from the central region of the wounds were stained with hematoxylin and eosin and their morphometric analysis was performed as described by Schatteman et al. [11, 13]. Briefly, hematoxylin and eosin images of wound sections were analyzed with Image-Pro Plus by tracing the wound boundary that was considered as the presence of intact hair follicles and the transition from normal epidermis to hypertrophic epidermis. Wound volume was calculated by interpolation from the wound areas measured in every 20th section, and expressed as a ratio of the wound volume at each time point divided by the original wound volume at time 0 [11, 13]. Seven to 15 animals per group were analyzed as a total of three independent experiments.
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sonimmuno.com) for 30 minutes and washed with PBS, after which slides were visualized by using avidin biotin complex (ABC)–peroxidase kit (Elite kit; Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com) and 3,3-diaminobenzidine tetrahydrochloride (Vector Laboratories). To detect the EPC incorporation into mouse vasculature, slides were first stained with mouse CD31 as described above. Subsequently, slides were incubated with human CD31 (DakoCytomation, Carpinteria, CA, http://www.dakocytomation.us) and then with biotinylated anti-mouse IgG (Jackson ImmunoResearch Laboratories, Inc.). Positive immunoreactivity was visualized by using ABC-alkaline phosphatase kit (Vector Laboratories) and Vector Blue (Vector Laboratories). Controls of immunostainings were prepared with incubations with irrelevant class- and species-matched IgG. To detect EPC migration, wounds were placed in prelabeled base molds with frozen-tissue matrix (OCT; Sakura Finetek, Inc., Torrance, CA, http://www.sakura-americas.com) and were immediately snap-frozen in isopentane solution cooled in liquid nitrogen, before they were immunostained with von Willebrand factor (vWF) antibody (DakoCytomation).
Enzyme-Linked Immunosorbent Assay The cytokine secretion from EPCs was evaluated by Quantikine human immunoassays (R&D Systems, Minneapolis, http://www. rndsystems.com) according to the manufacturer’s protocols. Equal numbers (1 × 105/ml) of EPCs, HDMECs, or fibroblasts were seeded and incubated in new culture medium for 24 hours before the medium was collected. Conditioned media were analyzed for MCP-1, MIP-1α, and PDGF-BB. The cell culture media did not contain measurable amounts of these cytokines. Serial dilutions of recombinant proteins were used as standards.
Statistical Analysis All data are presented as means ± SEM. One-way analysis of variance, followed by Bonferroni’s post hoc multiple comparison test, was used to determine differences between groups. A p value of less than .05 was considered statistically significant and the number of samples examined is indicated by n.
Results Immunohistochemistry For the analysis of monocyte/macrophage recruitment and capillary density, wound sections prepared as above were treated with 0.1% hydrogen peroxidase to quench the endogenous peroxidase activity. Then wound sections were immersed in PBS containing 10% normal goat serum and incubated overnight at 4°C with a specific antibody such as F4/80 (Abcam Ltd., Cambridge, U.K., http://www.abcam.com) or mouse CD31 (Pharmingen, Chicago, http://www.bdbiosciences.com/pharmingen). The slides were then incubated with biotinylated anti-rat IgG (Jackson Immuno Research Laboratories, Inc., West Grove, PA, http://www.jackwww.StemCells.com
EPC Characterization We isolated early EPCs from adult peripheral blood and characterized their endothelial phenotype, as previously described [9, 12]. EPCs were positive for both DiI-acLDL uptake and FITCulex-lectin binding. Fluorescence-activated cell-sorting analysis confirmed the endothelial phenotype of EPCs (vWF, 53.3% positive; and vascular/endothelial (VE)–cadherin, 47.1% positive) and staining with a nitric oxide (NO)–specific fluorescent probe (diamino-fluorescein-2 diacetate) confirmed the NO production characteristic of endothelial cells (data not shown).
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Effects of EPC Transplantation on Wound-Closure Rate and Wound Volume The effects of EPC transplantation on wound healing were evaluated in a full-thickness excisional wound model in nude mice and were compared with those of mature endothelial cell (HDMEC) transplantation. As shown in Figures 1A and 1B, EPC transplantation accelerated the rate of wound closure as early as day 3 after surgery, compared with that observed after HDMEC transplantation or PBS injection (EPC = 39.2% ± 3.3%, HDMEC = 25.3% ± 4.8%, PBS = 17.4% ± 3.8% on day 3; p < .05). This reduction in
EPC Transplantation for Dermal Wound Healing wound area was consistently observed until day 9 after wounding (EPC = 49.1% ± 1.9%, HDMEC = 34.8% ± 8.0%, PBS = 35.8% ± 6.8% at day 5; EPC = 73.5% ± 1.5%, HDMEC = 52.3% ± 3.5%, PBS = 49.5% ± 4.3% at day 7; EPC = 79.0% ± 1.8%, HDMEC = 66.2% ± 5.6%, PBS = 67.8% ± 4.6% at day 9). In accord with wound-closure rates, wound volumes were also significantly decreased in the EPC-transplanted group at day 7 when compared with those of HDMEC-transplanted or PBS control groups (Fig. 1C). Wound volumes in EPC-treated groups were reduced to 19.1% ± 1.5% of the original wound volume, whereas those in HDMEC-treated and PBS controls were 28.1% ± 2.3% and 33.6% ± 3.2% of original size, respectively. However, there was no significant difference in wound volume among these groups at day 14.
Effect of EPC Transplantation on Monocyte/Macrophage Recruitment To assess the wound-healing effects of EPC transplantation, the number of monocytes/macrophages in the EPC- or HDMECinjected skin was evaluated by immunohistochemistry using F4/80, a murine monocyte/macrophage marker [14]. As shown in Figure 2A, numerous monocytes/macrophages were frequently observed in the entire wound area. When the F4/80-positive cells were counted in each group at day 5 after wounding, EPC-injected wounds showed a markedly increased number of monocytes/macrophages compared with HDMEC- or PBSinjected wounds (Fig. 2B). This observation can be explained by our enzyme-linked immunosorbent assay (ELISA) results, which showed that EPCs strongly expressed various chemokines and cytokines (MCP-1 = 33.5 ± 1.6 ng/105 cells; MIP-1α = 88.5 ± 0.6 ng/105 cells; PDGF-BB = 213.8 ± 17.6 pg/105 cells), whereas both HDMECs and fibroblasts secreted these chemo-cytokines negligibly (Fig. 2C). MCP-1 and MIP-1α are two major chemoattractants for monocytes/macrophages and play a key role in macrophage infiltration in the early phase of wound healing [15]. Moreover, PDGF-BB has been known to play a central role throughout all stages of wound healing by promoting fibroblast proliferation, matrix production, and enhancing the formation of granulation tissue [16, 17].
Effects of EPC Transplantation on New Capillary Formation Figure 1. EPC transplantation facilitates dermal excisional wound healing. (A): Representative images show wound healing in mice treated with EPCs, HDMECs, or PBS. Wounds were photographed at the times indicated, from days 0–12. EPC transplantation accelerates the wound-closure rate. Scale bar = 2 mm. The wound-closure rate (B) and the wound volume (C) relative to that achieved by HDMEC transplantation or PBS injection (*p < .05). Wound-closure rate and wound volume were calculated as the ratio (percentage) of the openwound area and volume at each time point divided by the area at time 0. Data are means ± SEM (n = 7). Abbreviations: EPC, endothelial progenitor cell; HDMEC, human dermal microvascular endothelial cell; PBS, phosphate-buffered saline.
To investigate whether local transplantation of EPCs augments neovascularization at the site of injury, capillary density was measured by CD31 staining of wound sections retrieved at day 14. Representative photographs of CD31 staining in Figure 3A reveal that there were numerous newly formed capillaries in the EPC-treated group, but a lower number of capillaries in the PBSand HDMEC-treated groups. Quantitative analysis revealed that the capillary density (number of vessels/high power field) in the granulation tissue was almost twofold higher in the EPC-transplanted group than in the PBS- or HDMEC-treated groups (EPC
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= 52.1 ± 6.0, HDMEC = 23.2 ± 4.9, PBS = 27.6 ± 2.2; p < .05) (Fig. 3B). However, there was no significant difference between the PBS-treated group and HDEMC-transplanted groups.
EPC Incorporation into Newly Formed Vasculature
Figure 2. EPCs promote the accumulation of monocytes/macrophages in the wound by secreting chemo-cytokines. Wound sections removed 5 days after injury were immunohistochemically stained with F4/80, a murine monocyte/macrophage marker. (A): Representative images of F4/80 staining in EPC-, HDMEC-, PBS-treated wounds. A wound stained with control nonspecific antibody showed no positive cells. Scale bar = 100 μm. (B): The number of monocytes/macrophages in the wounded area was significantly increased in the EPC-treated group compared with those of HDMEC- or PBS-treated groups (*p < .01). Data are means ± SEM (n = 6). (C): EPCs secrete various wound healing–related chemo-cytokines, including MCP-1, MIP-1α, and PDGF-BB. Conditioned medium from EPCs was harvested and analyzed by enzyme-linked immunosorbent assay. Data are expressed as means ± SEM (n = 4), and asterisks indicate a significant difference from the values for HDMECs and fibroblasts (*p < .01). Abbreviations: EPC, endothelial progenitor cell; HDMEC, human dermal microvascular endothelial cell; MCP-1, monocyte chemotactic protein 1; MIP1α, macrophage inflammatory protein–1α; PBS, phosphate-buffered saline; PDGF-BB, platelet-derived growth factor.
Figure 3. The EPC-transplanted group showed a marked increase in capillary density in the granulation tissue compared with that of the HDMEC-transplanted group or PBS group. The capillary density in the wounded skin was measured by immunohistochemical staining with mouse CD31 in wound sections collected 14 days after injury. (A): Representative images of mouse CD31 staining (arrowheads) of EPC-, HDMEC-, or PBS-treated wounds. Scale bar = 100 μm. (B): Capillary density (indicated by the number of CD31-positive dermal vessels) in the wounded area was significantly increased in the EPCtreated group compared with the HDMEC- or PBS-treated groups (*p < .01). Data are means ± SEM (n = 6). Abbreviations: EPC, endothelial progenitor cell; HDMEC, human dermal microvascular endothelial cell; IgG, immunoglobulin G; PBS, phosphate-buffered saline.
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To examine the migration of intradermally injected EPCs to ischemic wounds in the skin, EPCs and HDMECs were prelabeled with red fluorescent DiI dye before injection and were tracked on frozen wound sections by fluorescence microscopy. As shown in Figures 4A–4E, HDMEC-treated groups had a few scattered DiI-labeled red fluorescent cells located in the granulation tissue under the scar at day 14 after injury. However, there were numerous red fluorescent cells in identical tissues from EPC-treated groups. The identity of red fluorescent cells in EPC-treated groups was confirmed by costaining with anti-vWF antibody, indicating that the red fluorescent cells were transplanted human EPCs (Figs. 4F–4H).
Figure 4. A number of endothelial progenitor cells (EPCs) were found in the dermis and hypodermis below the scar at day 14. Representative images under fluorescence microscopy show anatomic localization of (A) DiI (red)–labeled human EPCs and (B) human dermal microvascular endothelial cells in the wounded area stained with 4,6-diamidino-2-phenylindole (DAPI; blue) at day 14. (C–E): Closer examination of DiI-labeled human EPCs in wound sections. (F–H): Immunohistochemistry with human von Willebrand factor (vWF)–specific antibody (green) further confirmed the presence of EPCs. Scale bars = 100 μm.
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Wound sections were also examined for the presence of EPC incorporation into newly formed capillaries by immunostaining with human- and mouse-specific CD31 antibodies. As shown in Figure 5, blood vessels in the granulation tissue were frequently lined with both blue, human CD31–positive cells and brown, mouse CD31–positive cells, demonstrating that the injected EPCs were directly incorporated into the neovasculature during the wound-repair process. Quantitative examination of sections revealed that 7.5% ± 1.1% of neovessels were characterized to contain the injected human EPCs, and a majority of blood vessels was composed of host-derived cells. Although EPCs are directly involved in the formation of neovessels as a substrate of new endothelial cells, a major mechanism in the EPC-mediated neovascularization might be that EPCs promote endogenous angiogenesis in mouse by secreting angiogenic growth factors at EPC-incorporated foci, which in turn, contributes to the development of host-derived neovessels. In HDMEC-treated groups, there were few human CD31–positive cells associated with vessels (frequency is less than 0.1%). This result was confirmed by different immunolabeling experiments with fluorescent speciesspecific lectins (FITC-ulex-lectin and Alexa fluor 594–labeled Bandeiraea simplicifolia lectin B4 to detect human and mouse endothelial cells, respectively; data not shown). This observation indicates that HDMECs, fully differentiated endothelial cells, are not efficient either in migrating to ischemic tissues or in integrating to newly formed capillaries in granulation tissues, which was also observed by other researchers [4, 6].
Discussion With the recent discovery of EPCs in adult peripheral blood, a number of studies are under way to evaluate the neovascularization potential of EPCs in various animal models and several
Figure 5. Transplanted endothelial progenitor cells were directly incorporated into newly formed capillaries in granulation tissues. Immunohistochemistry was performed with human-specific CD31 (blue, arrowheads) and mouse-specific CD31 (brown) antibodies to differentiate the species origin of capillary endothelial cells. Scale bars = 20 μm.
EPC Transplantation for Dermal Wound Healing clinical trials [5, 18, 19]. EPC-mediated therapeutic vasculogenesis has been well illustrated in cardiovascular disease models in which circulating EPCs preferentially home to ischemic tissue and are directly incorporated into vessel walls, thereby contributing to the natural mechanisms of blood vessel formation. Similarly, EPC therapy should be very useful in wound repair, which requires a functional vasculature to carry a sufficient blood supply to meet the massive local demands for fibroblast proliferation, extracellular matrix synthesis, and re-epithelialization. In this study, a full-thickness excisional wound model was established in athymic nude mice to evaluate the wound-healing effects of EPC transplantation. When the neovascularization effects in the wounded tissues were analyzed, transplanted EPCs were observed to migrate from the injection site to the granulation tissue right below the scar. This EPC homing process is mainly regulated by VEGF and stromal-derived growth factor 1, which are highly upregulated in the ischemic areas of injured skin by hypoxia-inducible transcription [20, 21]. The recruited EPCs were directly incorporated into the neovasculature as endothelial precursors. As finding EPCs incorporated into capillaries was not a common event, enhanced neovascularization in EPC-treated groups might be explained with the finding that EPCs secrete high levels of various angiogenic cytokines, such as VEGF and PDGF-BB, which might activate adjacent endothelial cells and further facilitate the formation of new blood vessels in wounded tissues [6, 8, 9]. This paracrine effect of EPCs might also explain the accelerated rate of wound closure in the EPC-transplanted group compared with that of the HDMEC-transplanted control group. In particular, PDGF-BB has been reported to accelerate the re-epithelialization of open wounds in a series of animal and clinical studies [16, 22]. When PDGF-BB protein or DNA was applied on excisional full-thickness wounds, treated wounds closed faster than the controls, which appeared to occur through the promotion of re-epithelialization. This rapid wound closure might result from the fact that PDGF-BB stimulates fibroblasts to produce the extracellular matrix, subsequently establishing better substrate surface for keratinocyte migration. However, the direct effect of PDGF-BB on migration and proliferation of keratinocytes remains unclear due to the controversy over the expression of PDGF receptors on epidermal keratinocytes [23, 24]. Together with re-epithelialization and angiogenesis, inflammation plays an important role in wound healing, especially in the early phase. During this period, monocytes/macrophages remove bacteria and cell debris, and act as a source of inflammatory and growth-promoting substances, thereby enhancing the proliferation and differentiation of various cells within the wound. The functional importance of monocytes/macrophages in wound healing has been substantiated by many in vivo studies. For example, local injection of additional macrophages into wounds promoted the repair response in aged mice in which
Suh, Kim, Kim et al. wound healing is impaired [25]. The application of macrophageactivating substances also augmented the wound-repair process [26, 27]. Given the prominent roles of monocytes/macrophages in wound healing, the high level of infiltration of monocytes/ macrophages in EPC-treated wounds might increase the supply of cytokines and growth factors in the wounded tissue, resulting in the substantially accelerated wound closure observed in the EPC-transplanted group. The recruitment of monocytes/macrophages is tightly regulated by CC chemokines, such as MCP1 (CCL2), MIP-1α, -1β (CCL3, CCL4), and RANTES (CCL5) [15, 28]. The monocyte/macrophage chemoattractant activities of these chemokines are evident in that treatment with their neutralizing antibodies causes a decrease in the number of macrophages at the wound site, suggesting a direct involvement of these chemokines in the infiltration of monocytes/macrophage [28, 29]. Therefore, the increased infiltration of monocytes/ macrophages in EPC-transplanted tissue might be explained by the considerable amounts of MCP-1 and MIP-1α produced endogenously by EPCs, as shown by our ELISA data. However, further research is required to understand the precise mechanisms by which transplanted EPCs facilitate the recruitment of monocytes/macrophages into the wound.
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of monocytes/macrophages in early wound sections. These findings suggest that improved wound healing by EPC transplantation might be mediated through abundant monocyte/macrophage recruitment, as well as by increased neovascularization. EPC-assisted wound healing is potentially a therapeutic approach for the treatment of chronic wounds, in which natural wound-healing processes are insufficient to prevent tissue necrosis and ischemia, partially because of an insufficient secretion of growth factors and inadequate circulation of oxygenated blood. Although many attempts have been made to improve chronic wounds by administering angiogenic growth factors such as VEGF, clinical results have been discouraging, with only modest improvements in the length of time to closure, in breaking strength, and in neuropathy [30–32]. However, EPC therapy has several theoretical advantages over growth factor–mediated approaches, in that transplanted EPCs not only act as endothelial substrates in the formation of new blood vessels, but also provide cytokines and growth factors important for wound healing. Furthermore, EPCs home to injured tissues and exert their effects in those areas most in need of new blood vessel growth. Therefore, EPC transplantation may be regarded as an attractive therapeutic option for the treatment of chronic wounds, which remain a major clinical problem, especially in diabetic patients.
Conclusion We have demonstrated that EPC transplantation accelerates cutaneous wound repair in a murine dermal excisional wound model. Transplanted EPCs were directly involved in the formation of new capillaries in the granulation tissue, thereby promoting neovascularization relative to that of the control mice transplanted with HDMEC. Furthermore, the EPCs used in this study were shown to produce high levels of various chemo-cytokines (MCP-1, MIP-α, and PDGF-BB), which may explain the high degree of infiltration
Acknowledgments
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This work was supported by National Research Laboratory grants from the Korea Institute of Science and Technology Evaluation and Planning (M1-0203-00-0048) to D.-K.K. W.S. and K.L.K. contributed equally to this article.
Disclosures The authors indicate no potential conflicts of interest.
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See www.StemCells.com for supplemental material available online.
