RAPID COMMUNICATION Mesenchymal stromal cells prevent bleomycin-induced lung and skin fibrosis in aged mice and restore wound healing† Gustavo A. Rubio1, Sharon J. Elliot1, Tongyu Wikramanayake2, Xiaomei Xia3, Simone Pereira-Simon1, Seth R. Thaller1, George D. Glinos2, Ivan Jozic2, Penelope Hirt2, Irena Pastar2, Marjana Tomic-Canic2*, Marilyn K. Glassberg.1,3* *jointly directed this study 1 DeWitt Daughtry Family Department of Surgery, University of Miami Leonard M. Miller School of Medicine, Miami, FL, USA. 2 Wound Healing and Regenerative Medicine Research Program, Department of Dermatology and Cutaneous Surgery, University of Miami Leonard M. Miller School of Medicine, Miami, FL, USA. 3 Department of Medicine, University of Miami Leonard M. Miller School of Medicine, Miami, FL, USA. Corresponding Authors: Marilyn K. Glassberg, M.D. Division of Department of Medicine, University of Miami Leonard M. Miller School of Medicine 1600 NW 10th Ave RMSB 7056 (D-60) Miami, FL 33136, USA Phone: +1-305-243-3709 E-mail: mglassbe@med.miami.edu Marjana Tomic-Canic, Ph.D. Department of Dermatology and Cutaneous Surgery University of Miami Leonard M. Miller School of Medicine 1600 NW 10th Ave RMSB 2023A Miami, FL 33136, USA E-mail: mtcanic@med.miami.edu †This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: [10.1002/jcp.26418] Additional Supporting Information may be found in the online version of this article. Received 19 October 2017; Accepted 19 December 2017 Journal of Cellular Physiology This article is protected by copyright. All rights reserved DOI 10.1002/jcp.26418 1 This article is protected by copyright. All rights reserved Abstract Fibrosis can develop in nearly any tissue leading to a wide range of chronic fibrotic diseases. However, current treatment options are limited. In this study, we utilized an established aged mouse model of bleomycin-induced lung fibrosis (BLM) to test our hypothesis that fibrosis may develop simultaneously in multiple organs by evaluating skin fibrosis and wound healing. Fibrosis was induced in lung in aged (18-22 month-old) C57BL/6 male mice by intratracheal BLM administration. Allogeneic adipose-derived mesenchymal stromal cells (ASCs) or saline were injected intravenously 24 hours after BLM administration. Full thickness 8-mm punch wounds were performed 7 days later to study potential systemic anti-fibrotic and wound healing effects of intravenously delivered ASCs. Mice developed lung and skin fibrosis as well as delayed wound closure. Moreover, we observed similar changes in the expression of known profibrotic factors in both lung and skin wound tissue, including microRNA-199 and protein expression of its corresponding target, caveolin-1, as well as phosphorylation of protein kinase B. Importantly, ASC-treated mice exhibited attenuation of BLM-induced lung and skin fibrosis and accelerated wound healing, suggesting that ASCs may prime injured tissues and prevent endorgan fibrosis. This article is protected by copyright. All rights reserved 2 This article is protected by copyright. All rights reserved INTRODUCTION Aberrant wound healing resulting in fibrosis can occur in nearly all tissues and organ systems leading to a wide variety of chronic diseases that together account for nearly a third of worldwide disease-related mortality (Rockey et al., 2015; Wynn, 2008; Zeisberg and Kalluri, 2013). Despite representing a major global health issue, few, if any, effective anti-fibrotic therapies are available to date. Thus, there is a great clinical need to better understand the mechanisms involved in fibrosis and to develop effective and safe therapies. This aberrant fibrogenic process is thought to involve common cellular and molecular pathways (Rockey et al., 2015; Zeisberg and Kalluri, 2013). Such pathways may be involved in fibrotic diseases in multiple organs and, therefore, represent an attractive target for novel anti-fibrotic therapies. The fibrogenic cascade may be initiated by a variety of stimuli resulting in tissue injury such as infections, autoimmune reactions, radiation, or chemical injury (Wynn, 2008). Furthermore, aging plays an important role and is one of the prominent contributing factors. In response to injury, a complex inflammatory reaction ensues that under normal conditions is adaptive and results in normal wound healing (Rockey et al., 2015). In certain disease states and in aging, a phenotypic reprogramming may occur in cells leading to an imbalance between extracellular matrix (ECM) deposition and resorption, resulting in tissue fibrosis (Rockey et al., 2015; Usunier et al., 2014; Wynn, 2008; Zeisberg and Kalluri, 2013). A goal of anti-fibrotic therapies is, therefore, to restore an “acute repair phenotype" in fibrotic tissues to promote healing and tissue restoration. 3 This article is protected by copyright. All rights reserved Appropriate pre-clinical models are necessary to study potential systemic anti-fibrotic therapies that may target common pathways and have beneficial effects in multiple tissues. Among established animal models of fibrosis, bleomycin (BLM) has been used to induce lung and skin fibrosis (Ishikawa et al., 2009; Marangoni et al., 2016; Tashiro et al., 2015; Wu and Varga, 2008). BLM causes chelation of metal ions and oxygen free radical production leading to DNA breaks and tissue injury resulting in fibrosis (Moeller et al., 2008). Rodent models of intratracheal BLM-induced lung fibrosis have been implemented to study idiopathic pulmonary fibrosis (IPF) (Moeller et al., 2008; Peng et al., 2013). Additionally, subdermal BLM injections have been used to induce skin fibrosis and mimic some of the manifestations of scleroderma (Ishikawa et al., 2009; Lee et al., 2014; Liang et al., 2015; Marangoni et al., 2016). Furthermore, BLM can be used to induce injury to multiple organs, as demonstrated with repeated high doses of subcutaneous BLM causing skin, lung, and gastrointestinal injury (Ishikawa et al., 2009). Therefore, BLM injury resulting in skin and lung fibrosis may provide a pre-clinical model to study potential systemic anti-fibrotic therapies that target multiple organs. Among potential systemic anti-fibrotic therapies under investigation, mesenchymal stem/stromal cells (MSCs) have emerged as one of the most promising. MSCs have been shown to exert anti-inflammatory, anti-oxidant, and immunomodulatory effects, as well as an ability to modulate pro-fibrotic factors (Eming et al., 2014; Toonkel et al., 2013; Usunier et al., 2014). MSCs have been investigated in pre-clinical and clinical studies for diseases in various organs including the heart (Chen et al., 2016), lung (Glassberg et al., 2016), kidney (Wang et al., 2016), liver (Haldar et al., 2016) and skin (Ojeh et al., 2015; Otero-Vinas and Falanga, 2016). Benefits of MSCs in various organs are thought to stem from their ability to adapt to the local environment and regulate its secretome (Usunier et al., 2014). Thus, one potential advantage of 4 This article is protected by copyright. All rights reserved MSCs, in contrast to individual anti-fibrotic drugs, is their ability to act on several pathways simultaneously and orchestrate a phenotypic reprogramming. One such way that MSCs may exert anti-fibrotic effects and restore an “acute repair phenotype” is by modulating several pathways simultaneously via delivery of microRNAs (Baglio et al., 2015; Wang et al., 2016). In this study, we utilized an established aged mouse model of BLM-induced, nonreversible, lung fibrosis (Tashiro et al., 2015) to test our hypothesis that fibrogenesis may develop simultaneously in multiple organs by evaluating skin fibrosis and its impact on wound healing. Furthermore, we utilized this model to evaluate the potential of systemic allogeneic MSC administration to restore an “acute repair phenotype”, attenuate fibrosis and promote simultaneous cutaneous wound healing and lung repair by modulating pro-fibrotic factors. We induced pulmonary fibrosis in aged (18-22 month-old) male C57BL/6 by intratracheal BLM followed by full thickness 8-mm punch biopsy wounds 7 days later, coinciding with the onset of pulmonary fibrosis (Izbicki et al., 2002). Allogeneic adipose-derived mesenchymal stromal cells (ASCs) or saline were injected intravenously 24 hours after BLM administration. At 14-days post-wounding, lungs and cutaneous wound tissue were collected for functional and molecular assessments. We demonstrate that mice subjected to BLM-induced lung fibrosis also exhibited skin fibrosis and delayed wound closure. Moreover, we demonstrated similar changes in lung and skin wound tissue in the expression of known pro-fibrotic factors, including microRNA-199 and corresponding protein expression of its target, caveolin-1 (CAV-1), as well as phosphorylation of protein kinase B (AKT), all of which have been implicated in molecular pathways of fibrosis (Castello-Cros et al., 2011; Lino Cardenas et al., 2013; Mercer et al., 2016b). Lastly, ASC-treated mice exhibited attenuation of BLM-induced lung and skin fibrosis 5 This article is protected by copyright. All rights reserved and accelerated wound healing, suggesting that ASCs may prime injured tissues and prevent the end-organ fibrosis. METHODS Aged murine model of BLM-induced lung injury and delayed wound healing: All experiments were performed in accordance with approved guidelines and regulations. All experiments and procedures were approved by the Institutional Animal Care and Use Committee at the Leonard M. Miller School of Medicine at the University of Miami (Miami, FL), a facility accredited by the American Association for the Accreditation of Laboratory Animal Care (protocol 13-131). Male C57BL/6 mice were obtained from Jackson Laboratories (Bar Harbor, ME). Aged (18 to 22-month old) male mice were used for all experiments (n= 6/group). ASCs were isolated from young (4-month old) male C57BL/6 donors (Tashiro et al., 2015). Animals were housed under pathogen-free conditions with food and water ad libitum. BLM-induced lung injury: Pulmonary fibrosis was induced by intratracheal BLM administrated as previously reported (Tashiro et al., 2015). In brief, mice were anesthetized with ketamine (200 mg/kg) and xylazine (10 mg/kg) injected intraperitoneally, followed by direct intratracheal instillation of bleomycin sulfate (Sigma-Aldrich Corp; St. Louis, MO) dissolved in 50 μl sterile saline (2.0 units per kg of bodyweight). Control mice received 50 μl of intratracheal sterile saline. One day following BLM or saline admnistration, mice were treated with either ASC or saline intravenous injection as described below. Wounding: Mice were wounded on day 7 after BLM or saline administration. Mice were again anesthetized with ketamine and xylazine injected intraperitoneally. Hair on the dorsal skin was removed with a clipper, and the skin was cleaned with providone-iodine antiseptic solution. A 6 This article is protected by copyright. All rights reserved circular full-thickness wound was induced along the dorsal midline with an 8-mm punch, and covered with transparent film dressing (Tegaderm™, 3M, St. Louis, MO) for 24 hours, then exposed to room air. Mice were monitored daily and were euthanized 14 days later (21 days after BLM/saline treatment) and their lungs and skin wound tissue were harvested for analyses. Wound tissue and skin on the dorsal midline away from the wound sites were collected and bisected along the dorsal midline. Half of the specimens were fixed in 10% phosphate-buffered formalin and the other half were snap frozen for RNA and protein analyses. ASC isolation from young male donors: Donor ASCs were isolated from the subcutaneous adipose pads of 4-month old male C57BL/6 mice, as previously described (Tashiro et al., 2015). Briefly, subcutaneous adipose tissue was excised, washed in phosphate buffer solution (PBS) without Ca2+ and Mg2+ (PBS) containing 30% GIBCO® Pen/Strep (Life Technologies; Grand Island, NY) and digested in media containing 0.75% type II collagenase (Sigma-Aldrich; St. Louis, MO). Adipocytes were separated from the stromal vascular fraction by centrifugation. Resultant pellet was resuspended and cultured in ADSC™ Growth Medium (Lonza Group Ltd; Basel, Switzerland) and expanded in plastic Thermo Scientific™ Nunc™ Cell Culture Treated Flasks with Filter Caps (Thermo Fisher Scientific, Inc., Waltham, MA). Cells were incubated for 24 hours, at which point non-adherent cells were removed. Once cells reached confluency, they were trypsinized, expanded for 2-3 passages and cryopreserved in RecoveryTM Cell Culture Freezing Medium (Life Technologies). Characterization of ASCs was performed as previously described (Tashiro et al., 2015). Briefly, ASCs were incubated with fluorescence-labeled antibodies and analyzed by flowassisted cell sorting (FACS) Canto™ II (BD Biosciences; San Jose, CA). For mesenchymal differentiation potential, Mouse Mesenchymal Stem Cell Functional Identification Kit (R&D 7 This article is protected by copyright. All rights reserved Systems Inc.; Minneapolis, MN) was used according to the manufacturer’s instructions. Pluripotency was assessed via osteogenic and adipogenic differentiation.(Aguilar et al., 2009) ASC injections: Young donor-derived ASCs (passage 2 or 3) were thawed in a 37° C water bath and washed in PBS to remove the cell freezing solution prior to injection. ASCs were then passed through a 70 μm cell strainer to remove cell clumps. Cells were counted and resuspended in PBS immediately prior to injection. One day following BLM or saline administration, mice were administered 5 x 105 ASCs in 200μl of PBS by tail vein injection over 1 minute. Control mice received 200 μl of saline by tail-vein injection. Lung histological analysis and Ashcroft scoring Right lungs were inflated with 10% neutral buffered formalin (NBF) under 25 cm H2O constant pressure. Lungs were fixed in 10% NBF overnight and then transferred to PBS at 4°C. Samples were embedded in paraffin and 4 μm sections were taken for hematoxylin-eosin (H&E) and Masson’s Trichrome staining. Pulmonary fibrosis was assessed by a pathologist blinded to the experimental groups using the numerical Ashcroft scale (Ashcroft et al., 1988) on Masson’s Trichrome-stained slides at 20x magnification. Individual fields were assessed by systematically moving over a 32-square grid; each field was assessed for severity of fibrosis and assigned a score of 0 (normal lung) to 8 (total fibrosis of the field). Histomorphometric analyses of skin fibrosis and acute wound healing Formalin-fixed samples from skin adjacent to wounds were embedded in paraffin and 6µm sections were stained with Picrosirius Red reagents following manufacturer’s protocols (Electron Microscopy Sciences, Hatfield, PA). Representative images were captured on a Nikon Eclipse E400 microscope with polarized light (Nikon Inc., Melville, NY). Formalin-fixed wound samples were also embedded in paraffin and 6-µm sections were taken for H&E and Masson 8 This article is protected by copyright. All rights reserved Trichrome staining. Representative images were captured on a Zeiss Observer D1 microscope (Carl Zeiss Microimaging Inc., Thornwood, NY). Total wound size (distance between normal connective tissues flanking the wound scar) and wound gap (distance between the migrating epithelial tongues) were measured and analyzed in each treatment group. Western blotting Lung and skin wound tissue were homogenized and lysates were collected for Western blot analyses, as previously described.(Tashiro et al.) Protein lysates were loaded onto 10% polyacrylamide gels for analysis of AKT (10 μg), pAKT (25 μg), and CAV-1 (10 μg) protein expression. Primary antibodies and concentrations used were as follows: goat polyclonal antiAKT1/2 N19 (1:1000) (Santa Cruz Biotechnology, Dallas, TX), rabbit polyclonal anti-pAKT S473 (1:1000) and rabbit polyclonal anti-caveolin-1 3238S (1:2000) (Cell Signaling Technology, Danvers, MA). Immunoreactive bands were determined by exposing nitrocellulose blots to a chemiluminescence solution (Denville Scientific Inc.; Metuchen, NJ) followed by exposure to Amersham Hyperfilm ECL (GE Healthcare Limited; Buckinghamshire, UK). Image J version 1.48v (National Institutes of Health; Bethesda, MD) was used to determine relative density of bands. β-actin expression was determined using mouse monoclonal anti-β-actin (1:10000) (Sigma-Aldrich, St. Louis, MO). All values were corrected for corresponding β-actin band. Real-time polymerase chain reaction (RT-PCR) RT-PCR was performed to determine the expression levels of miR-199-3p, as well as mRNA expression of αv-integrin and TNFα. Total RNA was extracted from lung and skin wound tissue homogenates. Amplification and measurement of target RNA was performed on the Step 1 real time PCR system, as previously described (Karl et al., 2006). TaqMan probes and primers for amplification of the specific transcripts were designed using the Primer Express 1.5 9 This article is protected by copyright. All rights reserved from Applied Biosystems (Foster City, CA). TaqMan ribosomal RNA control reagents (Life Technologies, Carlsbad, CA) designed to detect 18S ribosomal RNA, were used as an endogenous control. For miR-199-3p analysis, cDNA was generated using qScript™ microDNA cDNA Synthesis Kit (Quanta Biosciences, Beverly, MA) according to manufacturer’s instructions. Amplification of miR199-3p was performed using specific primers (IDT Biologika, Rockville, MD) using Real-Time SYBR Green qRT-PCR Amplification kit (Quanta Biosciences, Beverly, MA). U6 expression was used as a control for miR199 analysis, and relative expression was calculated using the comparative CT method.(Schmittgen and Livak) Statistical analysis All values are expressed as mean ± standard error of the mean (SEM). Overall significance of differences within experimental groups was determined by analysis of variance (ANOVA) in combination with Tukey’s multiple comparison test. Significance of differences between groups was determined using Student’s t-tests, with Welch’s correction as appropriate; p values less than 0.05 were considered statistically significant. RESULTS Single-dose intratracheal BLM-induced pulmonary fibrosis results in skin fibrosis and delayed wound healing in aged C57BL/6 mice It has previously been shown that BLM-induced pulmonary fibrosis is less severe and can spontaneously resolve in young mice, an effect not observed in aged mice (Redente et al., 2011; Sueblinvong et al., 2012). Therefore, in this study we used the previously established aged mouse model of BLM-induced pulmonary fibrosis (Tashiro et al., 2015) to assess whether these mice also develop skin fibrosis leading to wound healing impairment. Significant fibrosis was 10 This article is protected by copyright. All rights reserved observed by 21-day sacrifice in lung histological sections of BLM-treated mice compared to saline-treated controls (Fig. 1A). Ashcroft scores, a semi-quantitative measure of lung fibrosis on histological sections (Ashcroft et al., 1988), were significantly increased in BLM-treated compared to saline-treated group (Fig. 1B). Standard picrosirius red staining was used to analyze the systemic effects of BLM on collagen network alterations on intact dorsal (unwounded) skin. In saline-treated mice, collagen fibers were long and well-aligned in a normal orientation parallel to the epidermis (Fig. 1F). In BLM-treated mice, however, collagen fibers were shortened and misaligned, including areas where they were perpendicular to the epidermis (Fig. 1F), which is consistent with skin fibrosis (Vorstenbosch et al., 2013). Moreover, histomorphometric analyses of wound healing including total wound size (distance between normal connective tissues flanking the wound scar) and wound gap (distance between the migrating epithelial tongues) were measured using H&E stained sections 14-days post-wounding. BLM treatment resulted in impaired wound healing, compared to the saline-treated control mice that exhibited normal wound healing (Fig. 1C). Total wound size was approximately 3-fold larger in BLM-treated mice compared to saline-treated mice (Fig. 1D; p>0.01). Similarly, there was a 6-fold increase in the size of wound gap in BLM-treated mice relative to saline-treated mice (Fig. 1E; p<0.01). These results demonstrate a significant delay in wound healing in mice with BLM-induced lung injury. Single-dose intravenous ASC treatment prevents lung and skin fibrosis and accelerates wound healing in BLM-treated aged mice We have previously shown that allogeneic young male donor-derived ASCs can prevent the development of pulmonary fibrosis following intratracheal BLM instillation in aged mice (Tashiro et al., 2015). Given that ASCs appear to exert therapeutic effects in multiple organs and 11 This article is protected by copyright. All rights reserved may modulate common fibrogenic pathways, we hypothesized that systemic ASC treatment may also prevent skin fibrosis and enhance wound healing in mice with BLM-induced pulmonary fibrosis by restoring an “acute repair phenotype” in injured tissues. Similar to our previous study, intravenous ASC treatment prevented the development of BLM-induced lung fibrosis that was observed in saline-injected mice (Fig. 1A and 1B). ASC treatment also prevented the pathological effects of BLM on skin collagen fibers, as demonstrated by well-aligned collagen fibers parallel to the epidermis similar to the collagen network observed in saline-treated control mice (Fig. 1F). Furthermore, ASC treatment prevented an impairment in wound healing in mice subjected to BLM-injury (Fig. 1C), as evidenced by a significant reduction in the total wound size by 58.8% compared to wounds of saline-injected mice (Fig. 1D; p<0.01). Similarly, ASCtreated mice had a wound gap size 69.5% smaller compared to saline-treated mice (Fig. 1E; p<0.05). Profibrotic micro-RNA-199-3p expression is increased in lung and skin wounds of BLMtreated mice, an effect prevented by systemic ASC treatment MicroRNAs (miRs) are major regulators of gene expression that have been shown to target key genes in pro-fibrotic pathways common to several organs (Bowen et al., 2013). Thus, miR regulation represents an attractive approach to modulate pro-fibrotic pathways. Moreover, delivery of beneficial miRs has been proposed as a mechanism by which MSCs exert their antifibrotic effects (Wang et al., 2016). Among several pro-fibrotic miRs, miR-199-3p has been reported to be upregulated in lung, kidney, and liver fibrosis (Lino Cardenas et al., 2013). MiR199-3p has also been implicated in cutaneous wound healing response (Chan et al., 2012). Therefore, we examined miR-199-3p expression in lung and cutaneous wound tissue in our 12 This article is protected by copyright. All rights reserved study, and found an upregulation of miR-199-3p in both tissues (Fig. 2) in BLM-treated mice compared to saline controls (p<0.01). Mice treated with systemic ASCs following BLM-injury had lower expression of miR-199-3p in both skin wound tissue (Fig. 2A) and lungs (Fig. 2B) compared to saline-treated mice. CAV-1 protein expression downregulation in lung and skin wounds of BLM-treated mice was prevented by systemic ASC treatment Given the observed upregulation of miR-199-3p expression following BLM injury and its prevention by ASC treatment, we next sought to examine if protein expression of a confirmed target of miR-199-3p, CAV-1(Lino Cardenas et al., 2013), was also modulated. CAV-1 is an important structural molecule often involved in sequestering growth factor receptors and blocking their signaling from the plasma membrane. Moreover, CAV-1 is downregulated in fibrotic skin (Castello-Cros et al., 2011; Lino Cardenas et al., 2013) and lung diseases (CastelloCros et al., 2011). Western blot analysis showed that ASC treatment resulted in a prevention of the CAV-1 downregulation that was observed in skin and lungs of BLM-treated mice (Fig. 3). BLM-induced activation of AKT pathway in lung and skin wounds of aged mice was prevented by ASC treatment Phosphorylation of AKT has been implicated in the signaling pathway of fibrogenesis (Mercer et al., 2016a). As expected, Western blot analysis showed that levels of phospho-AKT were increased in lung and skin wounds of BLM-treated mice compared to saline-treated controls (Fig. 4). Importantly, this effect was prevented with systemic ASCs in both tissues in mice treated after intratracheal BLM administration (Fig. 4). 13 This article is protected by copyright. All rights reserved Molecular markers of inflammation and fibrosis are upregulated in lung and skin of aged mice by BLM, an effect prevented by ASC treatment Real-time polymerase chain reaction (RT-PCR) was used to detect expression of tumor necrosis factor alpha (TNFα) and αv-integrin. Results showed that treatment with ASCs prevented the significant upregulation of these markers associated with tissue inflammation and fibrosis in BLM-treated mice. TNF-α was increased in lung and skin wounds of BLM-treated mice compared to saline-treated controls indicating increased inflammation (Table 1; p<0.05). ASC treatment prevented BLM-induced upregulation of TNF-α expression in both lung and skin wound tissue (Table 1; p<0.05). Expression of αv-integrin mRNA, a transmembrane cell adhesion molecule that modulates tissue fibrosis(Conroy et al.), was also increased in lung and skin from BLM-treated mice compared to saline controls (Table 1; p<0.01 and p<0.05, respectively). Similarly, systemic ASC infusion resulted in prevention of αv-integrin mRNA upregulation in both lung and skin (Table 1; p<0.05). DISCUSSION Tissue fibrosis can affect nearly every organ system and represents a major global health challenge (Rockey et al., 2015). To date, there are limited treatment options to attenuate tissue fibrosis and prevent end-organ failure. Conditions such as aging and certain disease states can impair the normal tissue response to injury. This abnormal wound response may result in excessive ECM deposition leading to tissue fibrosis and healing impairment (Kapetanaki et al., 2013; Rockey et al., 2015). Common molecular pathways appear to be involved in this fibrogenic cascade that can affect multiple organs. Pre-clinical models of multi-tissue fibrosis 14 This article is protected by copyright. All rights reserved are, therefore, important to study novel therapies that target these common fibrotic pathways. In the present study, fibrosis was induced in lung and skin of aged mice by intratracheal BLM administration followed by wounding to study potential systemic anti-fibrotic and wound healing effects of cell-based therapy. Results suggest that systemic administration of allogeneic ASCs can restore an “acute repair phenotype” by modulating pro-fibrotic factors to prevent lung and skin fibrosis and promote wound healing. One of the novel aspects of this study is the use of an aged murine model of BLMinduced lung and skin fibrosis with impaired healing. Given the similarities of aberrant healing and fibrogenic pathways, such as in patients with IPF and chronic venous leg ulcers (Blumberg et al., 2012; Eming et al., 2014), we postulated that BLM-induced lung fibrosis would result in dysregulation of repair mechanisms leading to fibrosis in skin tissue and impaired wound healing. BLM is a chemotherapeutic agent that can cause oxygen free radical production and DNA breaks that can result in tissue fibrosis (Moeller et al., 2008). This drug has been widely used to induce lung fibrosis in pre-clinical animal models (Moeller et al., 2008; Peng et al., 2013). Previous studies have also reported induction of both lung and skin fibrosis by repetitive or continuous subdermal administration of BLM (Lee et al., 2014; Liang et al., 2015). These studies suggest that BLM can result in tissue injury and fibrosis at the local site of administration and has systemic effects that can promote fibrosis in other tissues. However, these studies have used young mice, which, in contrast to aged mice, spontaneously recover from BLM-induced fibrosis (Redente et al., 2011; Tashiro et al., 2015). Moreover, to our knowledge, this is the first report of a murine model of simultaneous lung and skin fibrosis, as well as impaired wound healing, after a single-dose of intratracheal BLM. This model, thus, may facilitate investigation of anti-fibrotic therapies that can be applicable to patients with fibrotic conditions. 15 This article is protected by copyright. All rights reserved Fibrosis is the end-result of a complex cascade of events that involves dysregulation in multiple pathways and inflammatory effector cells (Wynn, 2008). It is unlikely, therefore, that drugs that target single molecules or pathways will lead to effective anti-fibrotic therapies (Wynn, 2011). Given the complexity of fibrosis and intricacies of the end-organ tissues it is particularly challenging to develop a single drug therapy that can simultaneously reverse fibrosis in multiple organs. Also, combination therapies may be less effective given the potential of additive side effects. Cell-based therapies, such as ASCs, have been studied as anti-fibrotic therapy in several organs (Chen et al., 2016; Glassberg et al., 2016; Haldar et al., 2016; OteroVinas and Falanga, 2016; Wang et al., 2016). ASCs appear to act by paracrine effects to modulate multiple pathways and demonstrate an ability to adapt to the local tissue to restore homeostasis and promote tissue repair (Usunier et al., 2014). In the current study, aged mice were treated with intravenous allogeneic ASCs one day following intratracheal BLM injury. Results suggest that systemic treatment with ASCs prevented BLM-induced lung and dermal fibrosis, and enhanced wound healing in aged mice. This suggests that ASCs may have the ability to systemically target common molecular pathways involved in fibrosis in different organs. It is tempting to speculate that in addition to aiding in tissue repair, ASCs given on day 1 may prime the end-organ tissues for prompt acute repair resulting in protective effects in this aged mouse model. One mechanism by which ASCs may systemically regulate common fibrotic pathways is via modulation of miR expression in target tissues (Baglio et al., 2015; Wang et al., 2016). MiRs are small non-coding RNAs involved in post-transcriptional regulation of nearly 60% of all protein-encoding genes (Friedman et al., 2009; Pastar et al., 2012). Dysregulation of miRs has been implicated in fibrotic conditions including chronic skin wounds (Pastar et al., 2012) and 16 This article is protected by copyright. All rights reserved lung fibrosis (Pandit and Milosevic, 2015). Overlap in multiple miRs dysregulated in different fibrotic conditions suggests that therapies that modulate their expression may be effective in reversing common fibrotic pathways and promoting tissue repair. In our study, we observed that the pro-fibrotic miR-199 was upregulated in both lung and skin wound tissue in BLM-treated mice. Notably, systemic ASC therapy prevented this miR-199 upregulation in both tissues. MiR199 has been previously shown to be a key effector of fibrosis in multiple tissues including lung, kidney, and liver, via modulation of TGF-β signaling pathway and downregulation of CAV-1 expression (Lino Cardenas et al., 2013). CAV-1 is a structural protein found in the caveolae of the plasma membrane (Castello-Cros et al., 2011). It is a bone fide target of miR-199 (Lino Cardenas et al., 2013) and has been shown to be a key regulator of fibrosis in lungs (Drab et al., 2001; Wang et al., 2006; Yamaguchi et al., 2011) and skin (Castello-Cros et al., 2011). Importantly, induction of miR-199 and suppression of CAV-1 expression have also been reported in lungs of IPF patients (Lino Cardenas et al., 2013; Wang et al., 2006). In the current study, changes in CAV-1 expression paralleled those of miR-199 in both skin and lung, with a downregulation in response to BLM that was prevented by ASC treatment. These results are consistent with previous studies that have shown a downregulation of CAV-1 expression in response to BLM-induced lung fibrosis in mice (Lino Cardenas et al., 2013; Wang et al., 2006). Thus, results suggest that ASCs may prevent BLM-induced lung and skin fibrosis and promote wound healing by modulating the expression of miRs and its corresponding targets, which are involved in the fibrogenic pathway. Other signaling pathways and effectors that are dysregulated in fibrosis were also evaluated and paralleled the results above. Activation of the AKT signaling pathway has been shown to be involved in tissue fibrosis (Mercer et al., 2016b; Russo et al., 2011). In addition, we previously 17 This article is protected by copyright. All rights reserved showed that ASC treatment can prevent its activation in BLM-induced fibrotic lungs in aged mice (Tashiro et al., 2015). In the current study, AKT activation was increased in both skin and lung in BLM-treated mice, but this effect was prevented by ASC treatment. PI3K/AKT signaling pathway has been proposed as a potential therapeutic target in fibrotic conditions such as IPF (Mercer et al., 2016b), and these data support the claim that ASCs may, in part, alter the course of pulmonary fibrosis by modulating this pathway. Other markers of inflammation and fibrosis, αv-integrin and TNFα, were also upregulated in lung and skin wound in response to BLM, and this was prevented by ASC therapy. These results further support the concept that ASCs appear to modulate multiple pathways involved in aberrant wound healing and fibrosis. In summary, in the current study, lung and skin fibrosis was induced by a single intratracheal administration of BLM in aged mice, which also resulted in irreversible fibrosis and impaired wound healing. This pre-clinical model of fibrosis in two tissues may, therefore, be used to evaluate mechanism of action and effectiveness of novel systemic anti-fibrotic therapies. Moreover, results of this study support the hypothesis that ASC administration may promote a systemic acute repair phenotype to prevent fibrosis in multiple organs and enhance wound healing by modulating pro-fibrotic factors such as miR-199 and its downstream target, CAV 1. These results highlight the potential of ASCs as systemic anti-fibrotic treatment that may act on multiple pathways and have therapeutic effects in multiple organs. 18 This article is protected by copyright. All rights reserved REFERENCES Aguilar, S., C.J. Scotton, K. McNulty, E. Nye, G. Stamp, G. Laurent, D. Bonnet, and S.M. Janes. 2009. Bone marrow stem cells expressing keratinocyte growth factor via an inducible lentivirus protects against bleomycin-induced pulmonary fibrosis. PloS one. 4:e8013. Ashcroft, T., J.M. Simpson, and V. Timbrell. 1988. 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Common and organ-specific mechanisms associated with tissue fibrosis. Am J Physiol Cell Physiol. 304:C216-225. ACKNOWLEDGEMENT We are grateful to all members of our laboratories for helpful criticisms and overall support. This work was funded by NIH grants NR013881 and NR015649 (MT-C). The authors also thank the Lester and Sue Smith Foundation and the James S. Fauver Pulmonary Fibrosis Research Fund for their generous support in research funding. The authors’ contributions are as follows: SJE, IP, SRT, MTC, SRT and MKG conceived and designed the study. GAR, TW, XX, SPS, GDG, IJ, and PH performed experiments and data acquisition. GAR, SJE, TW, IP, MTC, and MKG performed data analysis and interpretation. GAR, SJE, SRT, IP, MTC, and MKG participated in manuscript writing and critical revisions. All authors read and approved final manuscript. Dr. Glassberg serves as a consultant expert on advisory boards for Genentech, Boehringer Ingelheim, Patara Pharma, and Bellerophon Therapeutics. All other authors have indicated no conflict of interest. 22 This article is protected by copyright. All rights reserved FIGURE LEGENDS Figure 1. Intratracheal bleomycin (BLM) results in lung and skin fibrosis, as well as delayed wound healing in aged mice, an effect prevented by systemic adipose-derived mesenchymal stromal cells (ASC) treatment. (A) Histological sections of lung tissue were stained with Masson’s-Trichrome as described in Materials and Methods. Representative photomicrographs at 2x (upper), 20x (left), and 40x (right) magnification of lung sections from saline-treated control mice, BLM-treated mice, and BLM + ASCs. (B) Degree of pulmonary fibrosis on histological sections was measured by semi-quantitative Ashcroft score. BLM instillation resulted in increased Ashcroft score compared to saline controls. Treatment with ASCs following BLM administration resulted in decreased Ashcroft score. (C) Representative H&E staining of cutaneous wounds 14 days after induction. Red arrows indicate margin, and blue arrows indicate the epithelialized edges of the migrating tongues. Significantly increased total wound size (D) and wound gap (E) were observed in BLM-treated mice compared with saline-treated controls, suggestive of delayed healing. This delayed healing was prevented with ASC treatment (D, E). (F) Representative picrosirius red staining of intact skin 21 days after BLM treatment. BLM treatment resulted in altered collagen fiber morphology compared to saline-treated controls, and this effect was prevented in ASC-treated mice. White arrows indicate misaligned fibers. Dotted line denote basement membrane. HF: hair follicle; SG: sebaceous gland. Data are graphed as mean ± standard error of the mean (n=6/group). *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. BLM, bleomycin. Figure 2. Profibrotic micro-RNA-199-3p expression is increased in lung and skin wounds of bleomycin-treated mice, an effect prevented by systemic ASC treatment. Expression of 23 This article is protected by copyright. All rights reserved microRNA-199a-3p in skin wound (A) and lungs (B) of aged C57BL/6 mice is increased in response to BLM treatment compared to saline controls. Treatment with allogeneic ASCs 1 day post-BLM infusion resulted in lower expression of microRNA199-3p, similar to that of salineonly controls. MicroRNA-199-3p expression was measured by reverse transcriptase polymerase chain reaction as described in Materials and Methods. U6 expression was used as a control. Data are graphed as mean ± standard error of the mean (n=6/group). *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. Figure 3. Caveolin (CAV)-1 protein expression downregulation in lung and skin wounds of BLM-treated mice was prevented by systemic ASC treatment. Western blots were performed on skin wound (A) and lung (B) tissue from aged C57BL/6 mice treated with saline, BLM, and BLM + ASC to measure protein expression of CAV-1. BLM resulted in downregulation of CAV-1 expression compared to saline-treated controls in both skin wound and lungs. ASC treatment resulted in a prevention of this BLM-induced CAV-1 downregulation. Inset shows a representative Western blot of 3 mice per treatment group. Full-length blots are presented in Supplementary Fig. S1. Data are graphed as mean ± standard error of the mean (n=6/group). *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. Figure 4. BLM-induced activation of AKT pathway in lung and skin wounds of aged mice was prevented by ASC treatment. Ratio of phosphorylated AKT to AKT protein expression in skin wound (A) and lung (B) tissue of subjects was quantified by Western blot. Aged C57BL/6 mice treated with intratracheal BLM demonstrated increased pAKT/AKT protein expression in both tissues compared to saline-treated controls. Mice treated with intravenous infusion of allogeneic ASCs 1 day following BLM administration demonstrated decreased expression of pAKT/AKT compared to BLM-only group. Inset shows a representative Western blot of 3 mice 24 This article is protected by copyright. All rights reserved per treatment group. Full-length blots are presented in Supplementary Fig. S2. Data are graphed as mean ± standard error of the mean (n=6/group). *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. 25 This article is protected by copyright. All rights reserved TABLES Table 1. Expression of markers of fibrosis and inflammation in mice with bleomycininduced lung injury and delayed wound healing. Group (n=6/group) αvintegrin mRNA/18S TNF-α mRNA/18S Skin wound Saline Bleomycin only Bleomycin+ASCs 0.0806 ± 0.0193 0.4322 ± 0.1215* 0.0526 ± 0.0140†† 1.641 ± 0.3802 32.71 ± 13.38* 1.352 ± 0.2375† 0.5343 ± 0.1569 8.714 ± 1.786 Lung Saline Bleomycin only 1.484 ± 0.2556 ** 15.22 ± 2.168* 0.4048 ± 0.2964† 5.701 ± 2.649† Bleomycin+ASCs * p<0.05 vs. saline; *p<0.01 vs. saline; † p<0.05 vs. BLM only; ††p<0.01 vs BLM only ASCs, adipose-derived mesenchymal stem cells 26 This article is protected by copyright. All rights reserved Figure 1 27 This article is protected by copyright. All rights reserved Figure 2 28 This article is protected by copyright. All rights reserved Figure 3 29 This article is protected by copyright. All rights reserved Figure 4 30 This article is protected by copyright. All rights reserved 本文献由“学霸图书馆-文献云下载”收集自网络，仅供学习交流使用。 学霸图书馆（www.xuebalib.com）是一个“整合众多图书馆数据库资源， 提供一站式文献检索和下载服务”的24 小时在线不限IP 图书馆。 图书馆致力于便利、促进学习与科研，提供最强文献下载服务。 图书馆导航： 图书馆首页 文献云下载 图书馆入口 外文数据库大全 疑难文献辅助工具