Accepted Manuscript Possible contribution of fibrocytes to increased type I collagen synthesis during the early stage of dermal wound repair in human skin Min Sun, Peiru Wang, Toru Okubo, Jeffrey S. Orringer, John J. Voorhees, Gary J. Fisher, Yong Li PII:
S0022-202X(17)32835-X
DOI:
10.1016/j.jid.2017.08.020
Reference:
JID 1043
To appear in:
The Journal of Investigative Dermatology
Received Date: 26 January 2017 Revised Date:
14 July 2017
Accepted Date: 1 August 2017
Please cite this article as: Sun M, Wang P, Okubo T, Orringer JS, Voorhees JJ, Fisher GJ, Li Y, Possible contribution of fibrocytes to increased type I collagen synthesis during the early stage of dermal wound repair in human skin, The Journal of Investigative Dermatology (2017), doi: 10.1016/j.jid.2017.08.020. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Possible contribution of fibrocytes to increased type I collagen synthesis during the early stage of dermal wound repair in human skin Min Sun1,2, Peiru Wang1, Toru Okubo1, Jeffrey S. Orringer1, John J. Voorhees1, Gary J. Fisher1, Yong Li1,* 1:
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Department of Dermatology, University of Michigan, Ann Arbor, Michigan, USA
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Department of Dermatology, Xiangya Hospital, Central South University, Changsha, China. All work was performed at University of Michigan, Ann Arbor, MI USA.
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*To whom correspondence should be addressed: Department of Dermatology, Med. Sci. I, R6447, 1150 W Medical Center Drive, Ann Arbor, MI 48109-5609; Telephone: 734763-1469; Fax: 734-647-0076; Email:
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Short title: Fibrocytes produce collagen in wound repair
Keywords: Fibrocyte, Type I Collagen, Human Wound Repair Abbreviations: type I collagen (COL1), type I procollagen (proCOL1), carbon dioxide (CO2), peripheral blood mononuclear cells (PBMC), laser capture microdissection (LCM)
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Regeneration of the dermal extracellular matrix (ECM), which is primarily composed of type I collagen (COL1) fibrils, is essential for effective wound repair. During the early stage of dermal repair, rapid and marked increase of COL1-producing cells are typically seen. Skin resident fibroblasts are believed to be the major source of these COL1producting cells. Another source could be fibrocytes, which are fibroblast-like cells that differentiate from circulating hematopoietic progenitors (CD45+) (Bucala, 2015). The hallmark of the fibrocyte phenotype is co-expression of CD45 and COL1. Although the exact lineage of fibrocyte progenitors remains to be defined, these progenitors are readily detectable. Approximately 0.25% of peripheral blood mononuclear cells (PBMC) can differentiate into fibrocytes in vitro within a few days (Pilling et al., 2009b). Evidence regarding homing of fibrocyte progenitors to sites of injuries and differentiating into a significant number of COL1-producing fibrocytes remains inconclusive. Reports that fibrocytes are present in murine skin wounds have not been consistently substantiated (Barisic-Dujmovic et al., 2010; Fathke et al., 2004; Higashiyama et al., 2011; Suga et al., 2014). Little is known regarding fibrocytes in human skin wounds (Yang et al., 2005). We have investigated the origins of COL1-producing cells in a model of partial-thickness human wounds, which commonly occur. We have established a well-controlled partialthickness wound model in humans using ablative carbon dioxide (CO2) laser, and have utilized this model for investigating mechanisms underlying wound repair in humans (Orringer et al., 2004; Orringer et al., 2012). The CO2 laser ablates the entire epidermis and adjacent superficial dermis, which triggers a robust repair response with well
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characterized inflammatory, proliferative, and remodeling phases. During the early stage of dermal remodeling, we have consistently seen marked increase of COL1producing cells and large populations of CD45+ cells associated with blood vessels in the upper dermis. This observation has prompted us to investigate whether CD45+ cells contribute to COL1 production during wound repair in human skin in vivo.
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All procedures involving human subjects were approved by the University of Michigan Institutional Review Board, and performed according to the Declaration of Helsinki protocols. All subjects provided written informed consent. CO2 laser treatment, procurement of skin samples, and analyses were performed as previously reported (Orringer et al., 2004), and described in Supplemental Materials. In brief, 5-mm square wounds were generated by CO2 laser on buttock. Full-thickness punch biopsies (4 mm diameter) were obtained from the center of wounds at one and three weeks after wounding, and from skin with mock laser treatment.
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We assessed COL1-producing cells by immunostaining of type I procollagen (proCOL1), which is the soluble precursor of mature COL1. The anti-procollagen antibody used in this study recognizes both intracellular and extracellular type I procollagen. Hematopoietic cells were assessed using immunostaining of CD45. As expected, there were greatly increased numbers of COL1-producing cells and CD45+ cells in the upper dermis, at one week after wounding (Figure 1a and Supplemental Figure 1). Areas of densely packed cells, which were associated with blood vessels (Supplemental Figure 2), comprised 27±5.8% (n=9) of total area of wounded dermis.
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Interestingly, a fraction of CD45+ cells were also positive for proCOL1, as shown in a representative high magnification image (Figure 1b), suggesting that these cells are fibrocytes. We found that 15±6.2% (n=8) of CD45+ cells appeared to express proCOL1. These CD45+/proCOL1+ cells accounted for 25.8% of total proCOL1+ cells at one week after wounding.
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In addition to CD45, fibrocytes have been reported to express myeloid antigens CD11b and/or CD13 (Bucala, 2015). We found that the majority of cells in CD45-enriched areas of the dermis expressed CD13 (Supplemental Figure 3), whereas expression of CD11b was not detected (data not shown). A small proportion of the CD13+ cells also expressed proCOL1, indicating that at least some fibrocytes identified in our study are CD13+. At three weeks after wounding, the abundance of proCOL1+ cells increased, and the area occupied by CD45+ cells was significantly reduced to 9.0±2.4% (n=9) (Fig. 1c), compared to one week after wounding. Expression of proCOL1 by CD45+ cells was not observed at three weeks after wounding, indicating that the presence of CD45 and proCOL1 double positive cells is restricted to the early stages of wound repair.
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We next determined COL1 mRNA expression in areas of the dermis that have high density of CD45+ cells, at one week after wounding. We used laser capture microdissection (LCM) to separately obtain these areas for analysis. We isolated RNA from the captured tissue, and quantified COL1 mRNA levels, using real time RT-PCR. We found that COL1 mRNA was readily detectable (n=6, data not shown), indicating that there are COL1-producing cells localized in areas containing high density of CD45+ cells. The above results are consistent with the conclusion that a small proportion of CD45+ cells express COL1, at one week after wounding. These COL1-producing CD45+ cells display the fibrocyte phenotype.
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To further substantiate the presence of fibrocytes, we isolated CD45+ cells, using CD45 antibody-conjugated magnetic microbeads, from non-wounded and wounded skin, one week and three weeks after wounding. The same numbers of CD45+ cells were used for RNA extraction and COL1 mRNA quantification. COL1 mRNA was detectable in CD45+ cells isolated from skin one week after wounding, but not in CD45+ cells from non-wounded skin (Table-1). In addition, COL1 mRNA was not detectable in CD45positive cells from skin three weeks after wounding. These gene expression data are consistent with the immunostaining results (Fig.1c) that fibrocytes (CD45+/proCOL1+) were detected at one week, but not at three weeks post wounding. These results further support the conclusion that fibrocytes are present primarily in the early stage of dermal repair.
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Finally, we prepared fibrocyte-enriched cultures from peripheral blood mononuclear cells (PBMC), for comparison to our results from human skin. For these studies, PBMC were cultured under conditions that promote fibrocyte survival and differentiation (Pilling et al., 2009a). After 10 days, all adherent cells in the cultures displayed fibroblast morphology. The COL1 mRNA levels of these adherent cells were similar to those of cultured human skin fibroblasts (data not shown). Compared to the cultured fibrocytes, CD45+ cells isolated from wounded skin expressed 10-fold lower COL1 mRNA levels (Table 1), consistent with the data described above that a small proportion of CD45+ cells (approximately 15%) display a fibrocyte phenotype (Fig. 1). Taken together, the present study identifies the presence of CD45+/COL1+ fibrocytes in human skin, during the early stage of dermal wound repair. These fibrocytes likely differentiated from circulating fibrocyte precursors homing to skin wounds. The mechanisms that control fibrocyte homing and differentiation and the relative contribution of fibrocytes to COL1 production during wound healing remain to be determined. Our results support the view that fibrocytes can home to human skin wounds and facilitate COL1 regeneration, which may benefit wound repair. It is of interest to investigate the role of fibrocytes and their potential clinical applications in wounds that are prone to poor healing, such as partial thickness wounds in lower extremities, which often occur in persons with diabetes and the elderly.
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Figure Legends
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Figure 1. A fraction of CD45+ cells express proCOL1 protein at one week after wounding in human skin. Double immunofluorescence staining of proCOL1 and CD45 were performed on frozen sections from (a) non-wounded, (a and b) one week, and (c) three weeks post wounding. Nuclei were stained by blue DAPI (4',6-diamidino-2phenylindole). (b) representative high magnification image of CD45+ cell rich area in the dermis, one week after wounding. Cells co-stained with CD45 and proCOL1 are indicated by arrows. Images are representative of nine independent experiments.
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Table 1.CD45+ cells isolated from human skin one week after wounding express COL1 mRNA. CD45+ cells were isolated by immunoaffinity chromatography, and analyzed for COL1 mRNA levels by real time RT-PCR. COL1 mRNA levels were normalized to mRNA levels of housekeeping gene 36B4 (internal control). Results are means + SEM, N=4-6.
Conflict of interest
Relative COL1 mRNA levels under the limit of detection 0.14±0.08 under the limit of detection under the limit of detection 1.4±0.18
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Source of CD45+ Cells Non-wounded human skin Human skin one week after wounding Human skin three week after wounding Human PBMC Cultured human fibrocytes differentiated from PBMC
The authors state no conflict of interest.
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Acknowledgement
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We thank Suzan Rehbine, LPN, for biopsy procurement, Diane Fiolek for graphics and administrative support. This research was in part supported by Dermatology Foundation Career Development Award (YL). References
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Fathke C, Wilson L, Hutter J, Kapoor V, Smith A, Hocking A, et al. (2004) Contribution of bone marrow-derived cells to skin: collagen deposition and wound repair. Stem Cells 22:812-22.
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Higashiyama R, Nakao S, Shibusawa Y, Ishikawa O, Moro T, Mikami K, et al. (2011) Differential contribution of dermal resident and bone marrow-derived cells to collagen production during wound healing and fibrogenesis in mice. J Invest Dermatol 131:52936.
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