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Department of Medicine, Samsung Medical Center, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Seoul, Korea
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Key Words. Endothelial progenitor cell ? Macrophage ? Monocyte ? Neovascularization ? Wound healing2 C7 z' N: h2 B; s
3 [- }0 e7 |$ t" ~5 G( Q( |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
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9 V6 j0 U, V1 M- f2 F' |ABSTRACT; q% M# ^3 f2 S! y4 b, r
! J4 F6 R+ I5 HCutaneous 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 . 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 healing, 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 . In the next angiogenesis phase, new blood vessels form in response to an increase in the production 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.
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' ?6 p! y% M6 k w; o6 @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 . 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 . 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)–1 . 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.% U: L8 N( c* Q/ A w
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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 . These results might demonstrate that the wound-healing 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 chemo-cytokine secretion and the extent of neovascularization.* J Z* w7 H: e0 `) ~
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MATERIALS AND METHODS- m) C H! ~6 W7 Q
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EPC Characterization
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+ `3 N V* E$ a* O) R1 I8 e8 sWe isolated early EPCs from adult peripheral blood and characterized their endothelial phenotype, as previously described . EPCs were positive for both DiI-acLDL uptake and FITC-ulex-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).3 L4 a8 a- i$ [) v4 x; g& {% U( b
& @! }# C$ Y. P$ [2 NEffects of EPC Transplantation on Wound-Closure Rate and Wound Volume
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' V+ l4 ^1 a' M" oThe 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
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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
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5 u& `2 A* G# W# _8 A) T9 oEffect of EPC Transplantation on Monocyte/Macrophage Recruitment# y; T& ^* x9 S5 H/ O) p3 u
( [9 F! `4 l. C; J, d: m! OTo assess the wound-healing effects of EPC transplantation, the number of monocytes/macrophages in the EPC- or HDMEC-injected skin was evaluated by immunohistochemistry using F4/80, a murine monocyte/macrophage marker . 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 PBS-injected 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-1 = 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-1 are two major chemoattractants for monocytes/macrophages and play a key role in macrophage infiltration in the early phase of wound healing . 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 .
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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
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Effects of EPC Transplantation on New Capillary Formation
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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 PBS- and 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 = 52.1 ± 6.0, HDMEC = 23.2 ± 4.9, PBS = 27.6 ± 2.2; p - x+ p4 U4 J7 r8 R# @7 ]! O2 C
- A8 q: w6 y( A3 A/ vFigure 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 EPC-treated group compared with the HDMEC- or PBS-treated groups (*p
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1 n0 z# o$ z: v$ v k# M1 @ aEPC Incorporation into Newly Formed Vasculature
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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).
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7 L/ n5 O$ ^$ Y$ V5 U& }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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( e3 C P/ c% q2 X+ dWound 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 species-specific 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 .
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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.1 z( c+ T( A) D3 [. K
9 ~8 }; l8 y( l/ ZDISCUSSION
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8 G# c6 l3 Q2 H) `" i9 YWe 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-, and PDGF-BB), which may explain the high degree of infiltration 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.
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- |) {1 Y p1 Z6 KEPC-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 . 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.
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ACKNOWLEDGMENTS' K0 j/ r- q$ t( ^+ y
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发表于 2015-6-8 18:09
