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• W2583161808 abstract "Flap necrosis is the most frequent postoperative complication encountered in reconstructive surgery. We elucidated whether adipose-derived stem cells (ADSCs) and their derivatives might induce neovascularization and protect skin flaps during ischemia/reperfusion (I/R) injury. Flaps were subjected to 3 hours of ischemia by ligating long thoracic vessels and then to blood reperfusion. Qtracker-labeled ADSCs, ADSCs in conditioned medium (ADSC-CM), or ADSC exosomes (ADSC-Exo) were injected into the flaps. These treatments led to significantly increased flap survival and capillary density compared with I/R on postoperative day 5. IL-6 levels in the cell lysates or in conditioned medium were significantly higher in ADSCs than in Hs68 fibroblasts. ADSC-CM and ADSC-Exo increased tube formation. This result was corroborated by a strong decrease in skin repair after adding IL-6–neutralizing antibodies or small interfering RNA for IL-6 ADSCs. ADSC transplantation also increased flap recovery in I/R injury of IL-6–knockout mice. IL-6 was secreted from ADSCs through signal transducer and activator of transcription phosphorylation, and then IL-6 stimulated angiogenesis and enhanced recovery after I/R injury by the classic signaling pathway. The mechanism of skin recovery includes the direct differentiation of ADSCs into endothelial cells and the indirect effect of IL-6 released from ADSCs. ADSC-CM and ADSC-Exo could be used as off-the-shelf products for this therapy. Flap necrosis is the most frequent postoperative complication encountered in reconstructive surgery. We elucidated whether adipose-derived stem cells (ADSCs) and their derivatives might induce neovascularization and protect skin flaps during ischemia/reperfusion (I/R) injury. Flaps were subjected to 3 hours of ischemia by ligating long thoracic vessels and then to blood reperfusion. Qtracker-labeled ADSCs, ADSCs in conditioned medium (ADSC-CM), or ADSC exosomes (ADSC-Exo) were injected into the flaps. These treatments led to significantly increased flap survival and capillary density compared with I/R on postoperative day 5. IL-6 levels in the cell lysates or in conditioned medium were significantly higher in ADSCs than in Hs68 fibroblasts. ADSC-CM and ADSC-Exo increased tube formation. This result was corroborated by a strong decrease in skin repair after adding IL-6–neutralizing antibodies or small interfering RNA for IL-6 ADSCs. ADSC transplantation also increased flap recovery in I/R injury of IL-6–knockout mice. IL-6 was secreted from ADSCs through signal transducer and activator of transcription phosphorylation, and then IL-6 stimulated angiogenesis and enhanced recovery after I/R injury by the classic signaling pathway. The mechanism of skin recovery includes the direct differentiation of ADSCs into endothelial cells and the indirect effect of IL-6 released from ADSCs. ADSC-CM and ADSC-Exo could be used as off-the-shelf products for this therapy. Skin flap transplantation is most widely used in plastic and reconstructive surgery (Wang et al., 2011Wang W.Z. Baynosa R.C. Zamboni W.A. Update on ischemia-reperfusion injury for the plastic surgeon: 2011.Plast Reconstr Surg. 2011; 128: 685e-692eCrossref PubMed Scopus (107) Google Scholar). The poor healing of skin flaps is a common postoperative problem. The main reason might be the deficient neovascularization and diminished levels of proangiogenic factor production. Thus, determining how to increase proangiogenic factor levels and augment angiogenesis plays an essential role in improving the survival of skin flaps and increasing the success rate of skin transplantation. Cell-based therapy with stem cells is a promising option for ischemia/reperfusion (I/R) injury (Lindroos et al., 2011Lindroos B. Suuronen R. Miettinen S. The potential of adipose stem cells in regenerative medicine.Stem Cell Rev. 2011; 7: 269-291Crossref PubMed Scopus (342) Google Scholar). Adipose-derived stem cells (ADSCs) are a putative stem cell population within the adipose stromal compartment that have multiple differentiation potentials (Tapp et al., 2009Tapp H. Hanley Jr., E.N. Patt J.C. Gruber H.E. Adipose-derived stem cells: characterization and current application in orthopaedic tissue repair.Exp Biol Med. 2009; 234: 1-9Crossref PubMed Scopus (214) Google Scholar, Zuk, 2013Zuk P. Adipose-derived stem cells in tissue regeneration: a review.ISRN Stem Cells. 2013; : 1-35Crossref Google Scholar). In addition, ADSCs can promote neovascularization in lower limb ischemia (Lee et al., 2012Lee H.C. An S.G. Lee H.W. Park J.S. Cha K.S. Hong T.J. et al.Safety and effect of adipose tissue-derived stem cell implantation in patients with critical limb ischemia: a pilot study.Circ J. 2012; 76: 1750-1760Crossref PubMed Scopus (199) Google Scholar), myocardial infarction (Yang et al., 2013Yang D. Wang W. Li L. Peng Y. Chen P. Huang H. et al.The relative contribution of paracine effect versus direct differentiation on adipose-derived stem cell transplantation mediated cardiac repair.PLOS ONE. 2013; 8: e59020Crossref PubMed Scopus (103) Google Scholar), acute kidney injury (Gao et al., 2012Gao J.S. Liu R.F. Wu J. Liu Z.Q. Li J.J. Zhou J. et al.The use of chitosan based hydrogel for enhancing the therapeutic benefits of adipose-derived MSCs for acute kidney injury.Biomaterials. 2012; 33: 3673-3681Crossref PubMed Scopus (97) Google Scholar), and wound healing (Nauta et al., 2013Nauta A. Seidel C. Deveza L. Montoro D. Grova M. Ko S.H. et al.Adipose-derived stromal cells overexpressing vascular endothelial growth factor accelerate mouse excisional wound healing.Mol Ther. 2013; 21: 445-455Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). ADSCs secrete several growth factors such as platelet-derived growth factor, transforming growth factor-β, and vascular endothelial growth factor, which are known to favor angiogenesis in wound healing (Kim et al., 2007Kim W.S. Park B.S. Sung J.H. Yang J.M. Park S.B. Kwak S.J. et al.Wound healing effect of adipose-derived stem cells: a critical role of secretory factors on human dermal fibroblasts.J Dermatol Sci. 2007; 48: 15-24Abstract Full Text Full Text PDF PubMed Scopus (675) Google Scholar, Rehman et al., 2004Rehman J. Traktuev D. Li J.L. Merfeld-Clauss S. Temm-Grove C.J. Bovenkerk J.E. et al.Secretion of angiogenic and antiapoptotic factors by human adipose stromal cells.Circulation. 2004; 109: 1292-1298Crossref PubMed Scopus (1812) Google Scholar). However, it is not clear whether application of ADSCs could improve I/R injury of skin flaps and the related mechanisms. IL-6 is a pleiotropic cytokine that has a broad spectrum of biological activities (Fan et al., 2008Fan Y. Ye J. Shen F. Zhu Y. Yeghiazarians Y. Zhu W. et al.Interleukin-6 stimulates circulating blood-derived endothelial progenitor cell angiogenesis in vitro.J Cerebr Blood F Met. 2008; 28: 90-98Crossref PubMed Scopus (175) Google Scholar, Middleton et al., 2014Middleton K. Jones J. Lwin Z. Coward J.I. Interleukin-6: an angiogenic target in solid tumours.Crit Rev Oncol Hemat. 2014; 89: 129-139Crossref PubMed Scopus (103) Google Scholar). Although IL-6 has been implicated in wound healing and angiogenesis in peripheral tissue (Gallucci et al., 2000Gallucci R.M. Simeonova P.P. Matheson J.M. Kommineni C. Guriel J.L. Sugawara T. et al.Impaired cutaneous wound healing in interleukin-6-deficient and immunosuppressed mice.FASEB J. 2000; 14: 2525-2531Crossref PubMed Scopus (329) Google Scholar, Lin et al., 2003Lin Z.Q. Kondo T. Ishida Y. Takayasu T. Mukaida N. Essential involvement of IL-6 in the skin wound-healing process as evidenced by delayed wound healing in IL-6-deficient mice.J Leukocyte Biol. 2003; 73: 713-721Crossref PubMed Scopus (405) Google Scholar), the effects of IL-6 after I/R injury of skin flaps have not been investigated. Mesenchymal stem cells (MSCs) are promising parent cells that release exosomes (MSC-Exo) to repair damaged tissues (Han et al., 2016Han C. Sun X. Liu L. Jiang H.Y. Shen Y. Xu X.Y. et al.Exosomes and their therapeutic potentials of stem cells.Stem Cells Int. 2016; 2061: 1-11Google Scholar). However, the protein contents of exosomes released from ADSCs (ADSC-Exo) need further investigation to clarify their angiogenesis ability. Given these multiple functions, signaling of IL-6 is complex and can occur via two different pathways, classic and trans-signaling (Calabrese and Rose-John, 2014Calabrese L.H. Rose-John S. IL-6 biology: implications for clinical targeting in rheumatic disease.Nat Rev Rheumatol. 2014; 10: 720-727Crossref PubMed Scopus (233) Google Scholar, Jones et al., 2014Jones S.A. Fraser D.J. Fielding C.A. Jones G.W. Interleukin-6 in renal disease and therapy.Nephrol Dial Transpl. 2014; 30: 564-574Crossref PubMed Scopus (59) Google Scholar, Rose-John, 2012Rose-John S. IL-6 trans-signaling via the soluble IL-6 receptor: importance for the pro-inflammatory activities of IL-6.Int J Biol Sci. 2012; 8: 1237-1247Crossref PubMed Scopus (358) Google Scholar). In classic IL-6 signaling, IL-6 binds to membrane-bound IL-6 receptor (IL-6R), and then the complex of IL-6 and membrane-bound IL-6R associates with gp130, which initiates intracellular signaling. In trans–IL-6 signaling, a soluble form of IL-6R comprising the extracellular portion of the receptor can bind IL-6. The complex of IL-6 and soluble IL-6R can bind to gp130 on cells (Sakata et al., 2012Sakata H. Narasimhan P. Niizuma K. Maier C.M. Wakai T. Chan P.H. Interleukin 6-preconditioned neural stem cells reduce ischaemic injury in stroke mice.Brain. 2012; 135: 3298-3310Crossref PubMed Scopus (68) Google Scholar). The differential signaling pathway is involved in the effects of IL-6 from ADSCs on flap recovery, and angiogenesis remains unclear. In addition, regulation of IL-6 expression requires a complex array of intracellular signaling pathways involving signal transducer and activator of transcription (STAT)-3 and mitogen-activated protein kinases (Fischer and Hilfiker-Kleiner, 2007Fischer P. Hilfiker-Kleiner D. Survival pathways in hypertrophy and heart failure: the gp130-STAT axis.Basic Res Cardiol. 2007; 102: 393-411Crossref PubMed Scopus (134) Google Scholar, German et al., 2011German C.L. Sauer B.M. Howe C.L. The STAT3 beacon: IL-6 recurrently activates STAT 3 from endosomal structures.Exp Cell Res. 2011; 317: 1955-1969Crossref PubMed Scopus (27) Google Scholar). Little is known about IL-6 production in ADSCs and the mechanisms of these effects. In this study, we showed that IL-6 in ADSCs, ADSCs in conditioned medium (ADSC-CM), and ADSC-Exo increased angiogenesis and enhanced recovery from I/R injury in the long thoracic artery pattern of skin flaps in mice. We further showed that STAT3 phosphorylation is involved in IL-6 expression and angiogenesis. Based on these results, ADSC-secreted IL-6 may serve as a new conceptual target for flap therapy. The characterization of ADSCs is shown in the Supplementary Materials online (see Supplementary Figures S1–S6 online). To investigate the effects of ADSCs on I/R injury, a mouse model with an extended pectoral skin flap based on the right long thoracic vessels (Figure 1a) was used to evaluate the effects of ADSCs on the survival area and angiogenesis of I/R injury. The survival area in the I/R + ADSC group was much larger than that in the I/R group (Figure 1b and c). The histologic evaluation of skin flaps was conducted with hematoxylin and eosin staining (Figure 1d and e). The I/R + ADSC group had smaller areas of inflammation, more intact epithelialization, and many hair follicles compared with the I/R group. We used a modified histologic scoring system (Abramov et al., 2007Abramov Y. Golden B. Sullivan M. Botros S.M. Miller J.J. Alshahrour A. et al.Histologic characterization of vaginal vs. abdominal surgical wound healing in a rabbit model.Wound Repair Regen. 2007; 15: 80-86Crossref PubMed Scopus (180) Google Scholar) (see Supplementary Figure S7 online). The histologic scores in the I/R group were significantly lower than those in the sham group, whereas the ADSC treatment group had higher scores (Figure 1f). ADSCs labeled with Qtracker were observed under a fluorescent microscope (Figure 2a). Qtracker particles (red color) were present in the cytoplasm of ADSCs. ADSCs were mainly distributed in the area around the injection site of the skin flap (Figure 2b). ADSCs were found adjacent to the vessels and took part in capillary formation at higher magnification (Figure 2c–e). Some Qtracker-labeled ADSCs had CD31 immunoreactivity. We also found that expression of CD90, a stem cell marker, was co-localized with CD31 expression (Figure 2f). The number of microvessels in the I/R + ADSC group (16.3 ± 1.9) was clearly increased compared with the I/R group (5.8 ± 1.4) by anti-CD31 immunostaining (Figure 2g, h, and j). These findings suggest that ADSC transplantation increases neovascularization in the flaps. Next, we examined the effect of ADSCs on vascular tube formation using the Matrigel assay (BD Biosciences, San Jose, CA) with ADSC-CM. As shown in Figure 2i, ADSC-CM accelerated the assembling of endothelial cells into primitive vascular tube-like structures. Far fewer tubes were observed using CM from Hs68 fibroblasts (Hs68-CM), which are important MSCs that are often used for wound healing (You and Han, 2014You H.J. Han S.K. Cell therapy for wound healing.J Korean Med Sci. 2014; 29: 311-319Crossref PubMed Scopus (114) Google Scholar) (Figure 2i and k). Taken together, these results show that ADSCs and ADSC-CM are involved in angiogenesis and capillary tube formation. To investigate the mechanisms by which ADSCs cause an enhancement in flap survival, the human angiogenesis array was used to screen the molecules secreted from ADSC-CM. Antibody array blot images and the corresponding lists of the tested angiogenic factors are shown in Supplementary Figure S8 online. IL-6 was the most significantly changed angiogenic factor between ADSC-CM and Hs68-CM (see Supplementary Table S1 online). IL-6 expression was higher in ADSC-CM than in Hs68-CM (Figure 3a). In addition, IL-6 levels in either ADSC-CL or ADSC-CM were significantly higher than those in Hs68 by ELISA (Figure 3b). IL-6 expression was stronger in ADSCs than in Hs68 cells by immunofluorescent staining, and IL-6–positive cells were quantified (Figure 3c). Similarly, a Western blot analysis also confirmed the data (Figure 3d). A previous study showed that IL-6 is important for angiogenesis (Fan et al., 2008Fan Y. Ye J. Shen F. Zhu Y. Yeghiazarians Y. Zhu W. et al.Interleukin-6 stimulates circulating blood-derived endothelial progenitor cell angiogenesis in vitro.J Cerebr Blood F Met. 2008; 28: 90-98Crossref PubMed Scopus (175) Google Scholar). We therefore tested the effect of ADSC-CM on angiogenesis and examined whether the effect was attributable to IL-6. ADSC-CM significantly increased the tube formation compared with the control, and this effect was significantly reduced by the addition of anti–IL-6 antibody or CM from IL-6 small interfering RNA-transfected ADSCs (Figure 3e and f). Recombinant IL-6 increased the tube formation in a dose-dependent manner. A concentration of IL-6 greater than 75 pg/ml was sufficient to have a significant effect on tube formation (see Supplementary Figure S9 online). To explore whether IL-6 was involved in flap recovery, CM were collected from ADSCs treated with or without control IgG, a neutralizing anti–IL-6 antibody (anti–IL-6), transfection of small interfering RNA for IL-6, and transfection of a control scrambled oligonucleotide sequence. The I/R flaps were treated with these different CM. Both the anti–IL-6 antibody and IL-6–silencing groups had significantly decreased flap recovery compared with the IgG or the control scrambled oligonucleotide sequence treatment groups, respectively (Figure 4a and b). The I/R, anti–IL-6 antibody, and IL-6–silencing groups exhibited serious inflammatory areas, less epithelialization, and few hair follicles on hematoxylin and eosin staining (Figure 4c–e). We also observed that the microvessel density was lower in flaps with CM from the anti–IL-6 antibody or IL-6–silencing treatment (Figure 4f–h). In addition, IL-6 expression was very weak in these groups based on immunohistochemistry (see Supplementary Figure S10 online). Furthermore, IL-6–knockout (KO) mice were used to clarify the pivotal role of IL-6 in promoting flap recovery during I/R injury. The administration of ADSCs led to remarkably larger survival areas (Figure 4i and j). The IL-6–KO mice showed serious I/R injury in the skin flap on hematoxylin and eosin staining (Figure 4k). In contrast, the ADSC-treated group showed abundant collagen deposits, more intact epithelialization, and many hair follicles (Figure 4k–m). The skin flaps of IL-6–KO mice treated with ADSCs had an increased number of microvessels in response to I/R injury on anti-CD31 immunohistochemistry (Figure 4n–p). Taken together, these results show that IL-6 expression in ADSCs promoted flap recovery and angiogenesis. It has been shown that MSC-Exo contribute to their paracrine effects on angiogenesis, wound healing, tissue regeneration, and immunomodulation (Kapur and Katz, 2013Kapur S.K. Katz A.J. Review of the adipose derived stem cell secretome.Biochimie. 2013; 95: 2222-2228Crossref PubMed Scopus (227) Google Scholar). We hypothesized that ADSC-Exo would have therapeutic potential for skin flaps, especially because of their ability to release IL-6. ADSC-Exo display rounded and double-membraned structures with a size of approximately 60–200 nm by transmission electron microscopy (Figure 5a). The exosome yield of 1 × 106 ADSCs per day was 2.1 mg/ml of protein with the Bradford method. IL-6 levels in ADSC-Exo were significantly higher than those in ADSC-Exo–free CM by ELISA (Figure 5b). Immunoblot analyses showed that exosomal marker CD63 was present in ADSC-Exo but absent in cell lysates (Figure 5c). It is thought that the molecular composition of exosomes reflects the specialized functions of their original cells (Katsuda et al., 2013Katsuda T. Kosaka N. Takeshita F. Ochiya T. The therapeutic potential of mesenchymal stem cell-derived extracellular vesicles.Proteomics. 2013; 13: 1637-1653Crossref PubMed Scopus (299) Google Scholar). Thus, we next investigated whether ADSC-Exo contained IL-6. ADSC-Exo contained an abundance of IL-6 by Western blot analysis (Figure 5c). Mice were treated with ADSC-Exo to characterize the effects of these exosomes on flap recovery. We found that the flap recovery in the mice injected with ADSC-Exo was significantly greater compared with the mice treated with saline (Figures 5d and e). The ADSC-Exo–treated group showed a relatively small inflammatory area, much more epithelialization, and many hair follicles (Figure 5f and g). The histologic scores for the ADSC-Exo–treated group were significantly higher than those for the control group (Figure 5h). The number of microvessels in the ADSC-Exo group clearly increased compared with the I/R group by CD31 immunostaining (Figure 5i–k). In addition, significantly more tube-like structures formed in ADSC-Exo–treated endothelial cells than in control endothelial cells (Figure 5l and m). These results show that ADSC-Exo have abundant IL-6 and imply that these exosomes can increase angiogenesis to promote flap recovery after I/R injury. To explore the phosphorylation of STAT3 and mitogen-activated protein kinases involved in the mechanisms of IL-6 production and angiogenesis, we analyzed the phosphorylation levels of STAT3, extracellular signal–regulated kinase (ERK), c-Jun N-terminal kinase, and p38 by Western blot. We observed that the phosphorylation levels of ERK and STAT3 were higher in ADSCs than in fibroblasts, whereas the levels of phosphorylated (p-) c-Jun N-terminal kinase and p-p38 were not different (Figure 6a). To explore the interdependence of molecular signaling events initiated by ERK and STAT3 on ADSCs, we examined IL-6 production in ADSCs in the presence of ERK and STAT3 inhibitors. When ERK phosphorylation was inhibited using PD98095, we observed a decrease in p-ERK level, whereas the levels of IL-6 expression and p-STAT3 did not change (Figure 6b). However, when we inhibited STAT3 phosphorylation using stattic, we observed a significant decrease in IL-6 expression, yet ERK phosphorylation remained unchanged (Figure 6c). Moreover, the IL-6R inhibitor tocilizumab (TCZ) significantly reduced the phosphorylation of STAT3 and ERK, as well as IL-6 production (Figure 6d). This result was corroborated by the finding by ELISA that treating ADSCs with stattic or TCZ resulted in a significant decrease in IL-6 concentration (Figure 6e). Furthermore, we examined the influence of CM from PD98059-, stattic-, or TCZ-treated ADSCs on angiogenesis. Fewer capillary-like structures formed in the stattic- or TCZ-treated groups compared with the control group, whereas CM from the PD98059-treated group had no effect (Figure 6f and g). Taken together, these results indicate that STAT3, but not ERK, is essential for IL-6 production and angiogenesis. Next, we sought to identify the IL-6 signaling pathways that are involved in IL-6 expression. The sgp130 Fc protein, which specifically blocks IL-6 trans-signaling without affecting IL-6 classic signaling (Rose-John, 2012Rose-John S. IL-6 trans-signaling via the soluble IL-6 receptor: importance for the pro-inflammatory activities of IL-6.Int J Biol Sci. 2012; 8: 1237-1247Crossref PubMed Scopus (358) Google Scholar), did not affect IL-6 expression in cells and CM by Western blot and ELISA, respectively, whereas the co-treatment of sgp130 and TCZ reduced IL-6 expression (Figure 6h and i). IL-6R was present only in the cell lysate of ADSCs but not in ADSC-CM (Figure 6j). Furthermore, TCZ treatment showed serious I/R injury in the skin flap and had few microvessels compared with the I/R + ADSC group, whereas sgp130 had less effect on the protective effects of ADSC administration (Figure 6k). These data indicate that IL-6 production from ADSCs is mainly by STAT3 phosphorylation. The IL-6 from ADSCs that improved flap recovery after I/R injury was mainly by the classic signaling pathway, not by the trans-signaling pathway. In this study, we showed that ADSCs, ADSC-CM, and ADSC-Exo significantly enhance skin flap survival after I/R injury. The main finding was that functional IL-6 secreted from ADSCs was found in CM and exosomes. Furthermore, the IL-6–improved recovery was accomplished through increased angiogenesis and STAT3 activation. These results suggest that ADSCs may serve as a promising cell source for exosome-based skin flap survival treatments. ADSCs were easily obtained from liposuction aspirates and grown in vitro for use in diverse clinical applications, and such cells appear to pose fewer ethical issues than stem cells derived from other sources. Because ADSCs have low senescence levels and immunogenicity as well as pluripotent differentiation potential, ADSCs become a hot spot (Kim and Heo, 2014Kim E.H. Heo C.Y. Current applications of adipose-derived stem cells and their future perspectives.World J Stem Cells. 2014; 6: 65-68Crossref PubMed Google Scholar). ADSCs can accelerate the closure of dermal wounds on diabetic mice (Amos et al., 2010Amos P.J. Kapur S.K. Stapor P.C. Shang H.L. Bekiranov S. Khurgel M. et al.Human adipose-derived stromal cells accelerate diabetic wound healing: impact of cell formulation and delivery.Tissue Eng Pt A. 2010; 16: 1595-1606Crossref PubMed Scopus (164) Google Scholar). ADSCs were injected at the flap pedicle and improved viability of random pattern skin flaps (Lu et al., 2008Lu F. Mizuno H. Uysal C.A. Cai X.B. Ogawa R. Hyakusoku H. Improved viability of random pattern skin flaps through the use of adipose-derived stem cells.Plast Reconstr Surg. 2008; 121: 50-58Crossref PubMed Scopus (202) Google Scholar). ADSCs prevented I/R injury in dorsal cranial basal flaps and in inferior epigastric skin flaps (Uysal et al., 2009Uysal A.C. Mizuno H. Tobita M. Ogawa R. Hyakusoku H. The effect of adipose-derived stem cells on ischemia-reperfusion injury: immunohistochemical and ultrastructural evaluation.Plast Reconstr Surg. 2009; 124: 804-815Crossref PubMed Scopus (81) Google Scholar, Reichenberger et al., 2012Reichenberger M.A. Heimer S. Schaefer A. Lass U. Gebhard M.M. Germann G. et al.Adipose derived stem cells protect skin flaps against ischemia-reperfusion injury.Stem Cell Rev Rep. 2012; 8: 854-862Crossref PubMed Scopus (50) Google Scholar). Human ADSCs improved the viability of random pattern flaps in rabbits without immunological rejection (Gong et al., 2014Gong L.L. Wang C. Li Y.R. Sun Q.Z. Li G.Z. Wang D.R. Effects of human adipose-derived stem cells on the viability of rabbit random pattern flaps.Cytotherapy. 2014; 16: 496-507Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). In this study, we established pectoral skin flaps in mice near the long thoracic vessels after I/R injury. This model was simple and reliable, and it can be used as an alternative to the random pattern and the superficial epigastric models for experiments and training. Our data showed that the local application of ADSCs improved flap recovery after I/R injury and increased the number of CD31-stained vessels. We detected Qtracker-labeled cells incorporated into vascular structures, which suggests that endothelial formation from ADSCs occurred in vivo. Based on these results, we showed that ADSCs promoted skin flap survival by augmenting angiogenesis. The ability of stem cells to alter the tissue microenvironment via secretion of soluble factors might contribute to tissue repair, in addition to their capacity for differentiation (Phinney and Prockop, 2007Phinney D.G. Prockop D.J. Concise review: mesenchymal stem/multipotent stromal cells: the state of transdifferentiation and modes of tissue repair—current views.Stem Cells. 2007; 25: 2896-2902Crossref PubMed Scopus (1554) Google Scholar). IL-6 has a broad spectrum of biological activity relating to the regulation of angiogenesis and inflammation (Fan et al., 2008Fan Y. Ye J. Shen F. Zhu Y. Yeghiazarians Y. Zhu W. et al.Interleukin-6 stimulates circulating blood-derived endothelial progenitor cell angiogenesis in vitro.J Cerebr Blood F Met. 2008; 28: 90-98Crossref PubMed Scopus (175) Google Scholar, Middleton et al., 2014Middleton K. Jones J. Lwin Z. Coward J.I. Interleukin-6: an angiogenic target in solid tumours.Crit Rev Oncol Hemat. 2014; 89: 129-139Crossref PubMed Scopus (103) Google Scholar). The direct neuroprotective and neurotrophic actions are attributed to IL-6 (Suzuki et al., 2009Suzuki S. Tanaka K. Suzuki N. Ambivalent aspects of interleukin-6 in cerebral ischemia: inflammatory versus neurotrophic aspects.J Cerebr Blood F Met. 2009; 29: 464-479Crossref PubMed Scopus (181) Google Scholar). IL-6–KO mice displayed an impaired angiogenic response to brain ischemia with decreased density of microvessels (Gertz et al., 2012Gertz K. Kronenberg G. Kalin R.E. Baldinger T. Werner C. Balkaya M. et al.Essential role of interleukin-6 in post-stroke angiogenesis.Brain. 2012; 135: 1964-1980Crossref PubMed Scopus (144) Google Scholar). Endothelial cell-derived IL-6 induced angiogenesis (Neiva et al., 2014Neiva K.G. Warner K.A. Campos M.S. Zhang Z. Moren J. Danciu T.E. et al.Endothelial cell-derived interleukin-6 regulates tumor growth.BMC Cancer. 2014; 14: 99Crossref PubMed Scopus (24) Google Scholar). The intracerebroventricular administration of recombinant IL-6 reduced ischemic brain damage (Loddick et al., 1998Loddick S.A. Turnbull A.V. Rothwell N.J. Cerebral interleukin-6 is neuroprotective during permanent focal cerebral ischemia in the rat.J Cerebr Blood F Met. 1998; 18: 176-179Crossref PubMed Scopus (380) Google Scholar). Moreover, IL-6 secreted by ADSCs enhanced the proliferation of cardiomyocytes (Przybyt et al., 2013Przybyt E. Krenning G. Brinker M.G.L. Harmsen M.C. Adipose stromal cells primed with hypoxia and inflammation enhance cardiomyocyte proliferation rate in vitro through STAT3 and Erk1/2.J Transl Med. 2013; 11: 39Crossref PubMed Scopus (52) Google Scholar). In this study, we showed that ADSCs expressed higher levels of IL-6 than fibroblasts. A significantly high level of IL-6 was found in ADSC-CM and ADSC-Exo. Moreover, we showed that targeting IL-6 with neutralizing antibodies against IL-6 and silencing IL-6 in ADSCs had a measurable impact on tissue repair and angiogenesis. Furthermore, IL-6–KO mice displayed impaired recovery and a low angiogenic response after I/R injury of the skin flap. These results suggest that ADSC-CM exerted tissue repair through paracrine mechanisms involving IL-6. The benefit is that the application of the ADSC-CM rather than the cells themselves could circumvent the problem of retention in stem cell therapy. Exosomes may directly stimulate target cells through receptor-mediated interactions or may transfer from various bioactive molecules from the host cells to the recipient cells (Katsuda et al., 2013Katsuda T. Kosaka N. Takeshita F. Ochiya T. The therapeutic potential of mesenchymal stem cell-derived extracellular vesicles.Proteomics. 2013; 13: 1637-1653Crossref PubMed Scopus (299) Google Scholar). Extensive studies have shown the therapeutic effects of MSC-Exo on myocardial infarction, acute kidney injury, traumatic brain injury, and acute lung injury (Bian et al., 2014Bian S.Y. Zhang L.P. Duan L.F. Wang X. Min Y. Yu H.P. Extracellular vesicles derived from human bone marrow mesenchymal stem cells promote angiogenesis in a rat myocardial infarction model.J Mol Med. 2014; 92: 387-397Crossref PubMed Scopus (492) Google Scholar, Sun et al., 2011Sun C.K. Yen C.H. Lin Y.C. Tsai T.H. Chang L.T. Kao Y.H. et al.Autologous transplantation of adipose-derived mesenchymal stem cells markedly reduced acute ischemia-reperfusion lung injury in a rodent model.J Transl Med. 2011; 9: 118Crossref PubMed Scopus (114) Google Scholar, Zhang et al., 2015Zhang Y. Chopp M. Meng Y. Katakowski M. Xin H. Mahmood A. et al.Effect of exosomes derived from multipluripotent mesenchymal stromal cells on functional recovery and neurovascular plasticity in rats after traumatic brain injury.J Neurosurg. 2015; 122: 856-867Crossref PubMed Scopus (465) Google Scholar, Zhou et al., 2013Zhou Y. Xu H.T. Xu W.R. Wang B.Y. Wu H.Y. Tao Y. et al.Exosomes released by human umbilical cord mesenchymal stem cells protect against cisplatin-induced renal oxidative stress and apoptosis in vivo and in vitro.Stem Cell Res Ther. 2013; 4: 34Crossref PubMed Scopus (454) Google Scholar). However, to our knowledge, no study has focused on the therapeutic potential and the molecular content of ADSC-Exo for I/R injury of skin flaps. We showed that ADSC-Exo increased the vascular density and increased skin flap survival. In this study we showed that ADSC-Exo were rich in IL-6 and promoted angiogenesis via IL-6. The elevating IL-6 in rat orbitofrontal cortex with adenovirus-mediated gene delivery reversed a cognitive deficit (Donegan et al., 2014Donegan J.J. Girotti M. Weinberg M.S. Morilak D.A. A novel role for brain interleukin-6: facilitation of cognitive flexibility in rat orbitofrontal cortex.J Neurosci. 2014; 34: 953-962Crossref PubMed Scopus (57) Google Scholar). In this context, IL-6 delivery using ADSC-Exo has an advantage in patient safety because it does not require use of viral vectors. ADSC-Exo, to our knowledge previously unreported, contains functional IL-6, which suggests that there is a promising new approach to IL-6 delivery either peripherally and/or directly into the skin for flap survival treatment. IL-6 production involves activation of two major downstream signaling pathways: STAT and mitogen-activated protein kinases (Ahmed and Ivashkiv, 2000Ahmed S.T. Ivashkiv L.B. Inhibition of IL-6 and IL-10 signaling and Stat activation by inflammatory and stress pathways.J Immunol. 2000; 165: 5227-5237Crossref PubMed Scopus (116) Google Scholar). In this study we showed that the phosphorylation level of STAT3 and ERK were higher in ADSCs than in fibroblasts. Direct inhibition of p-STAT3 with stattic resulted in reduced IL-6 expression and secretion, as well as decreased tube formation. The role of STAT3 in IL-6 expression is supported by a previous report that the increased expression of STAT3-responsive genes and expression of IL-6 within satellite cells augments the repair of skeletal muscle (McKay et al., 2009McKay B.R. De Lisio M. Johnston A.P. O’Reilly C.E. Phillips S.M. Tarnopolsky M.A. et al.Association of interleukin-6 signalling with the muscle stem cell response following muscle-lengthening contractions in humans.PLOS ONE. 2009; 4: e6027Crossref PubMed Scopus (110) Google Scholar). Recent clinical therapies for stroke show a beneficial effect on the reduction of lesion sizes due to the activation of STAT3 and involvement of IL-6 (Gertz et al., 2012Gertz K. Kronenberg G. Kalin R.E. Baldinger T. Werner C. Balkaya M. et al.Essential role of interleukin-6 in post-stroke angiogenesis.Brain. 2012; 135: 1964-1980Crossref PubMed Scopus (144) Google Scholar). We also showed that the IL-6R inhibitor TCZ reduced the protective effects of ADSCs on I/R injury in the skin flap, whereas the IL-6 trans-signaling blocker sgp130 had less impact on the effects of ADSCs. This is consistent with a previous report that IL-6 trans-signaling is proinflammatory, whereas classic IL-6 signaling is needed for regenerative or anti-inflammatory activities of the cytokines (Stefan, 2012Stefan R.J. IL-6 trans-signaling via the soluble IL-6 receptor: importance for the pro-inflammatory activities of IL-6.Int J Biol Sci. 2012; 8: 1237-1247Crossref PubMed Scopus (631) Google Scholar). The outcome indicated that TCZ binds to the IL-6–binding site of IL-6R and competitively inhibits IL-6 signaling and activities (Shinriki et al., 2009Shinriki S. Jono H. Ota K. Ueda M. Kudo M. Ota T. et al.Humanized anti-interleukin-6 receptor antibody suppresses tumor angiogenesis and in vivo growth of human oral squamous cell carcinoma.Clin Cancer Res. 2009; 15: 5426-5434Crossref PubMed Scopus (138) Google Scholar). Collectively, these results show that IL-6 secreted from ADSCs through STAT3 phosphorylation and then IL-6 stimulated angiogenesis and enhanced recovery after I/R injury by the classic signaling pathway. In conclusion, our study shows that ADSC treatment and its secretory factor IL-6 are effective for enhancing skin flap recovery and angiogenesis after I/R injury. To our knowledge, this finding has not been previously reported. This mechanism of enhanced flap survival might occur not only from the differentiation of ADSCs into endothelial cells but also from the ability of ADSCs to produce IL-6. The clinical administration of ADSCs and their derivatives has just begun, and further studies will help translate our experimental results into a broad clinical application. Human ADSCs were donated after written informed consent from healthy adults. The procedure was approved by the Human Ethics Committee of Cathay General Hospital (GGH-P103021). All procedures involving experimental animals were performed in accordance with the guidelines for animal care of the National Taiwan University (No. 20150502). Male C57BL/6J mice and C57BL/6J-derived IL6–/– (B6.129S2-IL-6tm1kopf/J, IL-6–KO mice) mice were purchased from the Jackson Laboratory (Bar Harbor, ME). A pectoral skin flap (4 × 1 cm2) based over the right long thoracic vessels was raised as described previously (Figure 1a) (Miyamoto et al., 2008Miyamoto S. Takushima A. Okazaki M. Shiraishi T. Minabe T. Harii K. Free pectoral skin flap in the rat based on the long thoracic vessels: a new flap model for experimental study and microsurgical training.Ann Plast Surg. 2008; 61: 209-214Crossref PubMed Scopus (15) Google Scholar). Microvessel density in flap paraffin section was determined by using the endothelial cell marker CD31. The angiogenic molecules secreted by ADSCs and Hs68 cells were determined by the Human Angiogenesis Antibody Array (RayBiotech, Norcross, GA) according to the manufacturer’s instructions. ADSC-Exo was isolated from medium using ExoQuick-TC Exosome Precipitation Solution (System Biosciences, Mountain View, CA) per the manufacturer’s instructions. Western blot analysis of proteins in cell lysates, CM, and exosomes was conducted as described previously (Shen et al., 2016Shen W.C. Liang C.J. Huang T.M. Liu C.W. Wang S.H. Young G.H. et al.Indoxyl sulfate enhances IL-1beta-induced E-selectin expression in endothelial cells in acute kidney injury by the ROS/MAPKs/NFkappaB/AP-1 pathway.Arch Toxicol. 2016; 90: 2779-2792Crossref PubMed Scopus (40) Google Scholar). Data are presented as mean ± standard error of the mean for three to six separate experiments. All statistical analyses were performed with one-way analysis of variance and Fisher test. For all analysis, P < 0.05 was considered significant. An expanded Methods section is available in the Supplementary Materials. The authors state no conflict of interest. This work was supported by research grants from the Ministry of Science and Technology (NSC 102-2628-B-002-001-MY3), Taiwan. Download .pdf (1.59 MB) Help with pdf files Supplementary Data" @default.
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• W2583161808 title "Adipose-Derived Stem Cells Protect Skin Flaps against Ischemia/Reperfusion Injury via IL-6 Expression" @default.
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