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Human endothelial progenitor cells-derived exosomes accelerate cutaneous wound healing in diabetic rats by promoting endothelial function
Journal of Diabetes and Its Complications xxx (2016) xxx–xxx
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Journal of Diabetes and Its Complications j o u r n a l h o m e p a g e : W W W. J D C J O U R N A L . C O M
Human endothelial progenitor cells-derived exosomes accelerate cutaneous wound healing in diabetic rats by promoting endothelial function Xiaocong Li, Chunyu Jiang, Jungong Zhao ⁎ Department of Radiology, Shanghai Jiao Tong University Affiliated Sixth People's Hospital
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Article history: Received 29 March 2016 Received in revised form 4 May 2016 Accepted 8 May 2016 Available online xxxx Keywords: Endothelial progenitor cells Exosomes Diabetes Angiogenesis Wound healing
a b s t r a c t Aims: Wound healing is deeply dependent on neovascularization to restore blood flow. The neovascularization of endothelial progenitor cells (EPCs) through paracrine secretion has been reported in various tissue repair models. Exosomes, key components of cell paracrine mechanism, have been rarely reported in wound healing. Methods: Exosomes were isolated from the media of EPCs obtained from human umbilical cord blood. Diabetic rats wound model was established and treated with exosomes. The in vitro effects of exosomes on the proliferation, migration and angiogenic tubule formation of endothelial cells were investigated. Results: We revealed that human umbilical cord blood EPCs derived exosomes transplantation could accelerate cutaneous wound healing in diabetic rats. We also showed that exosomes enhanced the proliferation, migration and tube formation of vascular endothelial cells in vitro. Furthermore, we found that endothelial cells stimulated with these exosomes would increase expression of angiogenesis-related molecules, including FGF-1, VEGFA, VEGFR-2, ANG-1, E-selectin, CXCL-16, eNOS and IL-8. Conclusion: Taken together, our findings indicated that EPCs-derived exosomes facilitate wound healing by positively modulating vascular endothelial cells function. © 2016 Elsevier Inc. All rights reserved.
1. Introduction Diabetic ulcers represent a worldwide medical problem and a substantial societal and financial burden. Wound healing is deeply dependent on neovascularization to restore blood flow. The neovascularization of endothelial progenitor cells (EPCs) has been reported in various tissue repair models, such as myocardial infarction, stroke, and peripheral arterial diseases (King, Balaji, Keswani, & Crombleholme, 2014). However, the therapeutic potential of circulating EPCs in diabetic patients is not feasible because of the low number of cells in peripheral blood.
Competing interests The authors declare that they have no competing interests. Funding This study was supported by grants from the National Natural Science Foundation of China (No. 81271683). Authors’ contributions Each author has contributed in a substantial way to the work described in the manuscript and its preparation. XL and CJ collected the data and commented to the results. XL and JZ designed the study, analyzed the data and wrote the manuscript. All authors read and approved the final manuscript. ⁎ Corresponding author at: Departments of Radiology, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, 600 Yishan Rd, 200233, Shanghai, China. Tel.: +86 2164369181; fax: +86 2164701361. E-mail address: [email protected] (J. Zhao).
Studies have showed promising improvement following transplantation of EPCs in tissue repair models (King et al., 2014; Madonna & De Caterina, 2011). The local injection of EPCs into the excisional dermal wounds of mouse markedly accelerated wound healing and promoted neovascularization of granulation tissues (Suh et al., 2005). In diabetic patients with peripheral arterial disease, transplantation of mobilized peripheral blood mononuclear cells resulted in improvement with decreased rest pain, and increased pain-free walk time (Tateishi-Yuyama et al., 2002). However, the rate of incorporation of EPCs into newly formed vessels is low (Crosby et al., 2000). Recently, several groups have revealed that the stimulation of resident endothelial cells via paracrine mechanisms is more important than the direct differentiation into mature endothelial cells (Hagensen et al., 2012; Mitchell, Fujisawa, Newby, Mills, & Cruden, 2015), and exosomes play a major role in paracrine mechanism (Baglio, Pegtel, & Baldini, 2012; Dittmer & Leyh, 2014; Sahoo et al., 2011). Exosomes, small membrane particles (40–100 nm in diameter) originating from multivesicular bodies (MVBs), are generated from many cell types, and play key roles in the intercellular communication via the horizontal transfer of proteins, and RNAs to target cells (De Jong, Van Balkom, Schiffelers, Bouten, & Verhaar, 2014; Raposo & Stoorvogel, 2013). Therefore, we hypothesized that
http://dx.doi.org/10.1016/j.jdiacomp.2016.05.009 1056-8727/© 2016 Elsevier Inc. All rights reserved.
Please cite this article as: Li, X., et al., Human endothelial progenitor cells-derived exosomes accelerate cutaneous wound healing in diabetic rats by promoting endothelial function, Journal of Diabetes and Its Complications (2016), http://dx.doi.org/10.1016/j.jdiacomp.2016.05.009
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X. Li et al. / Journal of Diabetes and Its Complications xxx (2016) xxx–xxx
EPCs derived-exosomes (EPC-Exos) may also promote tissue repair during wound healing. In this study, we isolated and purified exosomes released from human umbilical cord blood (UCB)-derived EPCs and tested whether these exosomes could accelerate wound healing in diabetic rat models. Moreover, we investigated in vitro the effect of EPC-Exos on vascular endothelial cells proliferation, migration, tube formation and angiogenesis-related genes expression. 2. Materials and methods 2.1. Culture and characterization of EPCs derived from human umbilical cord blood The methods were performed as described previously (Li et al., 2016). Briefly, human UCB samples (approximately 80–100 mL) were collected from healthy newborns. This study sought and received informed consent from the parents of the infants, and was approved by the Institutional Review Board at Shanghai Six People's Hospital, Shanghai Jiaotong University. UCB was overlaid onto separation medium and centrifuged following the protocols. The mononuclear cells were placed into plates pre-coated with type I rat tail collagen and cultured in EGM-2MV (Lonza, Walkersville, MD, USA). After 48 hours, non-adherent cells were removed. The medium was changed daily for 7 days and then every other day until the first passage. Early-passage EPCs (p2–6) were used in the subsequent experiments. For immunocytochemistry, briefly, the cells were fixed and then incubated with primary antibodies overnight, followed by incubation with secondary antibodies for one hour. Isotype irrelevant antibodies were used as negative controls. Nuclei were stained with DAPI (Invitrogen, Grand Island, NY, USA) for 5 min. The cells were then washed three times and viewed using a fluorescence microscope (Leica AF6000). The antibodies, including mouse anti-human CD31 (1:100), rabbit anti-human CD34 (1:100), rabbit anti-human vWF(1:100), were obtained from Abcam (Cambridge, Cambs, UK). Fluorescent staining was used to detect the uptake of Dil-ac-LDL (Molecular Probes, Eugene, OR, USA) and binding of FITC-UEA-l (Sigma-Aldrich, St. Louis, MO). Briefly, the cells were incubated with Dil-ac-LDL (15 μg/mL) for 4 hours, and then stained with FITC-UEA-l (10 μg/mL) for one hour and with DAPI for 5 min. The cells were washed three times and analyzed under a fluorescence microscope (Leica AF6000). For flow cytometry, cells were fixed and then incubated with the following primary antibodies: FITC-conjugated mouse anti-human CD31, APC-conjugated mouse anti-human CD34, PE-conjugated mouse anti-human VEGFR-2, and FITC-conjugated mouse anti-human CD45. The antibodies were obtained from BD Biosciences (San Jose, CA, USA). Non-specific fluorescence was determined by incubation of similar cell aliquots with isotype-matched mouse monoclonal antibodies. The cells were washed with PBS and analyzed by using the Guava easyCyte™ Flow Cytometer (Millipore, Billerica, MA, USA). 2.2. Isolation and identification of exosomes released from human UCBderived EPCs The methods were performed as described previously (Li et al., 2016). Briefly, 80% confluent EPCs cultured for an additional 24 hours in EGM-2MV media deprived of FBS and supplemented with 1 × serum replacement solution (PeproTech, Rocky Hill, NJ, USA). The conditioned media of EPCs was obtained and centrifuged at 300 × g for 10 min and 2000 × g for 10 min at 4 °C to remove dead cells and cellular debris. Subsequently, the supernatant was filtered using a 0.22 μm filter sterilize Steritop TM (Millipore) to remove residual dead cells and cellular debris. Hereafter, the supernatant was centrifuged at 4000 × g at 4 °C to about 200 μL by ultra filtration in a
15 mL Amicon Ultra-15 Centrifugal Filter Unit (Millipore) and then centrifuged at 100,000 ×g for one hour at 4 °C to pellet the small vesicles. Exosomes were stored at − 80 °C or used for downstream experiments. The protein concentration of the exosomes was determined using the Micro Bicinchoninic Acid (BCA) Protein Assay Kit (Thermo Fisher, Waltham, MA, USA). Exosomes were visualized using a Hitachi H-7650 transmission electron microscope (Hitachi, Tokyo, Japan). Western blots were used to analyze the incorporation of each protein into exosomes. The antibodies against CD9 (1:300), CD63 (1:300) and CD81 (1:300), were obtained from Abcam. 2.3. Establishment of diabetic rats wound model and treatment All procedures were approved by the Animal Research Committee of the Sixth People's Hospital at the Shanghai Jiao Tong University. Male SD rats (250–300 g) were intravenously injected at a dose of 45 mg/kg of STZ (Sigma-Aldrich, St.Louis, USA). After four weeks, rats with fasting blood glucose levels higher than 16.67 mmol/L were considered diabetic and included for the experiments. Rats were anesthetized by intraperitoneal injection of 75 mg/kg pentobarbital, and after shaving a full thickness of the excision wound of 2.0 cm in length and 0.2 cm depth was created. Rats were randomly assigned to 2 different treatment groups, which were subcutaneously injected at wound sites with 100 μL PBS (control group) or EPC-Exos (100 μg) around the wounds at four sites. 2.4. Histological analysis The wound skin and surrounding skin were perfused for with heparinized saline and fixed by formalin. Five sections (5-μm thickness) were collected on glass microscopy slides. Hematoxylin and eosin (H&E) staining was performed according to standard protocols. Scar width (mm) meaning the junction gap between the normal dermis and dermis in the wound tissues was measured. A LEICA DM 4000B microscope and LEICA application suite 3.8 software were used to obtain and analyze images. 2.5. In vitro effects of EPC-Exos on vascular endothelial cells Vascular endothelial cells from the human microvascular endothelial cell line, HMEC-1 (Center for Disease Control and Prevention, Atlanta, GA) were cultured in MCDB131 cell culture media (Gibco BRL, Grand Island, NY, USA) supplemented with 10% FBS (Gibco BRL). A Cell Counting Kit-8 assay (CCK-8; Dojindo, Kyushu Island, Japan) was used to assess cell proliferation. HMECs were seeded onto 96-well plates with exosomes (100 μg/mL) or PBS. At day 1, 2, 3, 4 and 5, CCK-8 solution (10 μL/well) was added to the HMECs. The absorbance was measured at 450 nm using a microplate reader. The optical density (OD) values represented the survival/proliferation of HMECs. The migration effects of exosomes on cells were evaluated in scratch assays. HMECs were plated in plates, and the confluent monolayer was scratched using a p200 pipette tip (1 mL) of serum-free MCDB131 media supplemented with exosomes (100 μg/mL) or PBS was added. The cells were photographed after 4 hours (t = 4 hours) and 8 hours (t = 8 hours). Wound areas were measured using the Image-Pro Plus 6.0 software. The capillary-network formation was monitored by performing tube-formation assays. HMECs were seeded onto Matrigel-coated 96-well plates and incubated in serum-free MCDB131 media supplemented with exosomes (100 μg/mL) or an equal volume of PBS. The cells were incubated for 4 hours (t = 4 hours) and 8 hours (t = 8 hours). The number of the total branch points and tubule lengths in 5 randomly chosen fields were examined using an inverted microscope. EPCs were labeled with Vybrant DiO dye (Molecular Probes, Carlsbad, CA, USA), according to the manufacturer's protocol. Briefly,
Please cite this article as: Li, X., et al., Human endothelial progenitor cells-derived exosomes accelerate cutaneous wound healing in diabetic rats by promoting endothelial function, Journal of Diabetes and Its Complications (2016), http://dx.doi.org/10.1016/j.jdiacomp.2016.05.009
X. Li et al. / Journal of Diabetes and Its Complications xxx (2016) xxx–xxx
the cells were labeled for 15 min. The cell-labeled suspension was centrifuged and the supernatant was discarded. The cells cultured for an additional 24 hours in serum-free EGM-2 MV. Subsequently, the exosomes were isolated from the culture medium, and incubated with HMECs for 2 hours. The cells were then washed and analyzed under a fluorescence microscope (Leica AF6000). Influence of exosomes on the expression of angiogenesis-related genes in endothelial cells was evaluated with qRT-PCR. HMECs were seeded in plates and cultured in serum-free MCDB131 media containing exosomes (100 μg/mL) or PBS for 24 hours. qRT-PCR analysis was performed using an ABI PRISM®7900HT System with SYBR Premix ExTaq™ II (Takara Biotechnology, Japan). GAPDH was employed as the housekeeping gene for internal normalization.
2.6. Statistical analysis All of the experiments were performed at least three times. The data are shown as the means ± SEM. Differences were analyzed using an unpaired Student's t-test. P values b 0.05 were considered statistically significant.
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3. Results 3.1. Identification of human UCB-derived EPCs and EPC-Exos Immunostaining and flow cytometry analysis were used to detect the expressions of cell-specific antigens in the EPCs colonies. The results indicated that EPCs showed the typical endothelial-like cobblestone morphology and expressed endothelial lineage markers, such as CD31, vWF, and VEGFR-2, and were negative for the hematopoietic cell-specific marker CD45. Moreover, these cells also expressed CD34 (Fig. 1A–C), which showed their stem cells identity. Additionally, these cells were further tested by incorporating ac-LDL and binding endothelial-specific lectin UEA-1 (Fig. 1D), which are characteristic features of endothelial lineage cells. These studies confirmed that EPCs had been successfully isolated from the human UCB. In transmission electron microscopy experiments, we found that the majority of exosomes exhibited a round-shaped morphology, with a size ranging from 40 to 80 nm (Fig. 1E). And Western blotting analysis indicated these exosomes expressed characteristic exosomal surface marker proteins: CD9, CD63 and CD81 (Fig. 1F).
Fig. 1. Identification of human UCB-derived EPCs and EPC-Exos. (A) The UCB-derived adherent cells showed the typical endothelial-like cobblestone morphology. Scale bar: 100 μm. (B) Immunostaining revealed that the adherent cells were positive for CD31, vWF and CD34. Nuclei were counterstained by DAPI. Scale bar: 50 μm. (C) Flow cytometric analysis showed that the EPCs-like cells were positive for CD31, CD34, and VEGFR-2, but negative for the hematopoietic cell-specific marker CD45, indicating their EPCs identity. (D) The UCB-derived EPCs were further defined by their ability to bind FITC-UEA-l and uptake Dil-ac-LDL. Scale bar: 50 μm. (E) Morphology of EPC-Exos under a transmission electron microscopy. Scale bar: 50 nm. (F) Western blotting analysis of exosomal surface marker proteins (including CD9, CD63, and CD81) in EPC-Exos.
Please cite this article as: Li, X., et al., Human endothelial progenitor cells-derived exosomes accelerate cutaneous wound healing in diabetic rats by promoting endothelial function, Journal of Diabetes and Its Complications (2016), http://dx.doi.org/10.1016/j.jdiacomp.2016.05.009
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Fig. 2. EPC-Exos promote wound healing in diabetic rats. (A) Gross view and the effects of treatment with PBS or EPC-Exos on wound closure at 7 and 14 days. *P b 0.05, and EPC-Exos vs. control (n = 8 in each group). (B) H&E staining of wound sections following treatment with PBS or EPC-Exos at 14 days post-wounding. The double-headed arrows indicate the edges of the scar. *P b 0.05, and EPC-Exos vs. control (n = 8 in each group).
3.2. EPC-Exos promote wound healing in diabetic rats In the animal model of type 1 diabetes, we evaluated wound healing in 2 groups of rats that were treated with PBS (control group) or EPC-Exos around the wound sites. As shown in Fig. 2A, rats treated with EPC-Exos showed accelerated wound closure than the control groups at days 7 and 14. (day 7: 68.46 ± 6.3% vs. 24.35 ± 5.6%; day 14: 94.22 ± 5.1% vs. 75.56 ± 7.4%; n = 8; P b 0.05). In addition, the scar lengths were markedly reduced in the EPC-Exos group, compared to the control groups. These data suggested that EPC-Exos could use for the treatment of diabetic wound. 3.3. Internalized EPC-Exos functional assays and angiogenesis-related genes expression of vascular endothelial cells Furthermore, we investigated the function of exosomes for endothelial cells by using a series of in vitro assays. The effect of
EPC-Exos on the proliferation of vascular endothelial cells was determined by CCK-8 cell counting analysis, indicating that the exosomes treatment markedly enhanced the proliferation of HMECs (Fig. 3A). A wound healing assay was used to exam the effect of EPC-Exos on the migration of endothelial cells, showing that these exosomes significantly enhanced the motility of HMECs (Fig. 3B). The branch numbers and tube length were measured to assess the ability of HMECs to form tubules. Compared with the control group, all indicators that evaluated the tube formation capability were profoundly increased in HMECs stimulated with exosomes (Fig. 3C). Taken together, our in vitro experiments indicated that EPC-Exos could activate an angiogenic program in vascular endothelial cells. Fluorescence microscopy analysis showed the DiO-labeled EPC-Exos were taken up and transferred to the perinuclear region of endothelial cells (Fig. 3D), indicating that the EPC-Exos entered into the target cells, or that exosomes were internalized by vascular endothelial cells.
Please cite this article as: Li, X., et al., Human endothelial progenitor cells-derived exosomes accelerate cutaneous wound healing in diabetic rats by promoting endothelial function, Journal of Diabetes and Its Complications (2016), http://dx.doi.org/10.1016/j.jdiacomp.2016.05.009
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Fig. 3. Internalized EPC-Exos functional assays and angiogenesis-related genes expression of vascular endothelial cells. (A) EPC-Exos promoted human microvascular endothelial cells (HMECs) proliferation as analyzed by Cell Counting Kit-8 assay. *P b 0.05. (B) EPC-Exos stimulated the migration of HMECs. Scale bar: 250 μm. (C) The in vitro angiogenesis assay showed that EPC-Exos enhanced the tube formation of HMECs. Scale bar: 100 μm. (D) Fluorescent microscopy analysis of DiO-labeled EPC-Exos internalization in HMECs. The green-labeled exosomes were visible in the perinuclear region of HMECs. Scale bar: 50 μm. (E) qRT-PCR analysis of the expression levels of angiogenesis-related genes in HMECs stimulated with EPC-Exos or an equal volume of PBS buffer. *P b 0.05.
Please cite this article as: Li, X., et al., Human endothelial progenitor cells-derived exosomes accelerate cutaneous wound healing in diabetic rats by promoting endothelial function, Journal of Diabetes and Its Complications (2016), http://dx.doi.org/10.1016/j.jdiacomp.2016.05.009
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To test whether angiogenesis-related genes expression was altered by internalization of EPC-Exos, qRT-PCR analysis was performed. As shown in Fig. 3E, multiple critical pro-angiogenic genes were profoundly up-regulated by more than 4-fold after treatment with EPC-Exos, including endothelial fibroblast growth factor1 (FGF-1), nitric oxide synthase (eNOS), interleukin-8 (IL-8), angiopoietin-1 (ANG-1), E-selectin, vascular endothelial growth factor A (VEGFA), vascular endothelial growth factor receptor 2 (VEGFR-2), and chemokine ligand-16 (CXCL-16) showed notable up-regulation in endothelial cells stimulated with exosomes. In addition, exosomes-stimulated endothelial cells showed significant down-regulation of matrix metalloprotein-9 (MMP-9). These data indicated that EPC-Exos modulated the endothelial cells partly through the expressions of various angiogenesis-related genes. 4. Discussions Herein, we revealed that EPCs-derived exosomes transplantation could accelerate cutaneous wound healing in diabetic rats. We also showed that EPC-Exos enhanced the proliferation, migration and tube formation of vascular endothelial cells in vitro. Furthermore, we found that endothelial cells stimulated with these exosomes would increase expression of angiogenesis-related molecules. Therefore, our findings indicated that EPC-Exos facilitate wound healing by positively modulating vascular endothelial cells function. Recent studies have suggested that EPCs enhance endothelial cells regeneration by paracrine mechanism, but not by differentiation into mature endothelial cells (Baker et al., 2013; Kim et al., 2010). Exosomes are key components of paracrine secretion, and represent an important cell-cell communication by delivery of functional RNAs and proteins (Al-Nedawi, Meehan, & Rak, 2009; Zhu & Fan, 2011). Previous studies reported that human EPCs-derived exosomes do not express human leukocyte antigen class I and class II antigens (Cantaluppi et al., 2012), therefore, there are not overt immune reactions in our study. The further application of exosomes is promising without the potential risks of stem cell therapy, such as maldifferentiation and tumorigenesis. Actually, exosome therapeutic tools have been demonstrated in various disease models, such as reducing cardiomyocytes hypertrophy and apoptosis (Gu et al., 2014), enhancing neoangiogenesis of human pancreatic islets (Cantaluppi et al., 2012), and improving neovascularization in a hindlimb ischemia model (Ranghino et al., 2012). Exosomes derived from human induced pluripotent stem cell-derived mesenchymal stem cells could accelerate cutaneous wound healing, reduce scar widths, and enhance the proliferation and migration of human umbilical vein endothelial cells and human dermal fibroblasts in vitro (Zhang et al., 2015). Exosomes derived from human urine derived stem cells could promote endothelial cells' proliferation, and tube formation, and accelerate wound closure and re-epithelization in rats (Yuan, Guan, Zhang, Zhang, & Li, 2016). However, few investigations have aimed to discuss exosomes derived from EPCs in wound healing. Comparing with the low EPCs number in peripheral blood of diabetic patients, EPCs derived from UCB are easily obtained and present a great proliferative potential (Ingram et al., 2004). Therefore, in present study, we derived the EPCs from human UCB and confirmed that cells showed typical morphology and specific surface antigens. The transmission electron microscopy revealed the majority of exosomes were 60–80 nm in diameter and expressed specific exosomal markers, such as CD9, CD63 and CD81, which was consistent with previous reports (Ribeiro, Zhu, Millard, & Fan, 2013). As we known, exosome small size protects them from phagocytosis by the mononuclear cell system, and the membrane structure assists in easily passing their components across the recipient cell membrane. Therefore, exosomes are more convenient to bring and transfer proteins and mRNAs/microRNAs into target cells and consequently change target cells (Ribeiro et al., 2013). Our data
revealed that the EPCs-secreted exosomes up-regulated the expression of pro-angiogenesis molecules in vascular endothelial cells, including FGF-1, VEGFA, VEGFR-2, ANG-1, E-selectin, CXCL-16, eNOS and IL-8. These pro-angiogenesis molecules are critical for regulation of blood vessel development, by promoting proliferation, migration, and tube formation on endothelial cells (Wong & Crawford, 2013). However, the mRNA levels of MMP-9 were remarkably decreased in endothelial cells stimulated with EPCs-derived exosomes. MMP-9 seems to be implicated in increased inflammation because they are expressed mainly by neutrophils and macrophages, which are both important to the inflammatory response. Studies suggest that the high levels of MMP-9 predict poor wound healing (Liu et al., 2009), and the delivery of MMP-9-siRNA could reduce MMP-9 expression in skin fibroblast cells to accelerate wound healing (Li et al., 2014). 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Please cite this article as: Li, X., et al., Human endothelial progenitor cells-derived exosomes accelerate cutaneous wound healing in diabetic rats by promoting endothelial function, Journal of Diabetes and Its Complications (2016), http://dx.doi.org/10.1016/j.jdiacomp.2016.05.009
Contents lists available at ScienceDirect
Journal of Diabetes and Its Complications j o u r n a l h o m e p a g e : W W W. J D C J O U R N A L . C O M
Human endothelial progenitor cells-derived exosomes accelerate cutaneous wound healing in diabetic rats by promoting endothelial function Xiaocong Li, Chunyu Jiang, Jungong Zhao ⁎ Department of Radiology, Shanghai Jiao Tong University Affiliated Sixth People's Hospital
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Article history: Received 29 March 2016 Received in revised form 4 May 2016 Accepted 8 May 2016 Available online xxxx Keywords: Endothelial progenitor cells Exosomes Diabetes Angiogenesis Wound healing
a b s t r a c t Aims: Wound healing is deeply dependent on neovascularization to restore blood flow. The neovascularization of endothelial progenitor cells (EPCs) through paracrine secretion has been reported in various tissue repair models. Exosomes, key components of cell paracrine mechanism, have been rarely reported in wound healing. Methods: Exosomes were isolated from the media of EPCs obtained from human umbilical cord blood. Diabetic rats wound model was established and treated with exosomes. The in vitro effects of exosomes on the proliferation, migration and angiogenic tubule formation of endothelial cells were investigated. Results: We revealed that human umbilical cord blood EPCs derived exosomes transplantation could accelerate cutaneous wound healing in diabetic rats. We also showed that exosomes enhanced the proliferation, migration and tube formation of vascular endothelial cells in vitro. Furthermore, we found that endothelial cells stimulated with these exosomes would increase expression of angiogenesis-related molecules, including FGF-1, VEGFA, VEGFR-2, ANG-1, E-selectin, CXCL-16, eNOS and IL-8. Conclusion: Taken together, our findings indicated that EPCs-derived exosomes facilitate wound healing by positively modulating vascular endothelial cells function. © 2016 Elsevier Inc. All rights reserved.
1. Introduction Diabetic ulcers represent a worldwide medical problem and a substantial societal and financial burden. Wound healing is deeply dependent on neovascularization to restore blood flow. The neovascularization of endothelial progenitor cells (EPCs) has been reported in various tissue repair models, such as myocardial infarction, stroke, and peripheral arterial diseases (King, Balaji, Keswani, & Crombleholme, 2014). However, the therapeutic potential of circulating EPCs in diabetic patients is not feasible because of the low number of cells in peripheral blood.
Competing interests The authors declare that they have no competing interests. Funding This study was supported by grants from the National Natural Science Foundation of China (No. 81271683). Authors’ contributions Each author has contributed in a substantial way to the work described in the manuscript and its preparation. XL and CJ collected the data and commented to the results. XL and JZ designed the study, analyzed the data and wrote the manuscript. All authors read and approved the final manuscript. ⁎ Corresponding author at: Departments of Radiology, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, 600 Yishan Rd, 200233, Shanghai, China. Tel.: +86 2164369181; fax: +86 2164701361. E-mail address: [email protected] (J. Zhao).
Studies have showed promising improvement following transplantation of EPCs in tissue repair models (King et al., 2014; Madonna & De Caterina, 2011). The local injection of EPCs into the excisional dermal wounds of mouse markedly accelerated wound healing and promoted neovascularization of granulation tissues (Suh et al., 2005). In diabetic patients with peripheral arterial disease, transplantation of mobilized peripheral blood mononuclear cells resulted in improvement with decreased rest pain, and increased pain-free walk time (Tateishi-Yuyama et al., 2002). However, the rate of incorporation of EPCs into newly formed vessels is low (Crosby et al., 2000). Recently, several groups have revealed that the stimulation of resident endothelial cells via paracrine mechanisms is more important than the direct differentiation into mature endothelial cells (Hagensen et al., 2012; Mitchell, Fujisawa, Newby, Mills, & Cruden, 2015), and exosomes play a major role in paracrine mechanism (Baglio, Pegtel, & Baldini, 2012; Dittmer & Leyh, 2014; Sahoo et al., 2011). Exosomes, small membrane particles (40–100 nm in diameter) originating from multivesicular bodies (MVBs), are generated from many cell types, and play key roles in the intercellular communication via the horizontal transfer of proteins, and RNAs to target cells (De Jong, Van Balkom, Schiffelers, Bouten, & Verhaar, 2014; Raposo & Stoorvogel, 2013). Therefore, we hypothesized that
http://dx.doi.org/10.1016/j.jdiacomp.2016.05.009 1056-8727/© 2016 Elsevier Inc. All rights reserved.
Please cite this article as: Li, X., et al., Human endothelial progenitor cells-derived exosomes accelerate cutaneous wound healing in diabetic rats by promoting endothelial function, Journal of Diabetes and Its Complications (2016), http://dx.doi.org/10.1016/j.jdiacomp.2016.05.009
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EPCs derived-exosomes (EPC-Exos) may also promote tissue repair during wound healing. In this study, we isolated and purified exosomes released from human umbilical cord blood (UCB)-derived EPCs and tested whether these exosomes could accelerate wound healing in diabetic rat models. Moreover, we investigated in vitro the effect of EPC-Exos on vascular endothelial cells proliferation, migration, tube formation and angiogenesis-related genes expression. 2. Materials and methods 2.1. Culture and characterization of EPCs derived from human umbilical cord blood The methods were performed as described previously (Li et al., 2016). Briefly, human UCB samples (approximately 80–100 mL) were collected from healthy newborns. This study sought and received informed consent from the parents of the infants, and was approved by the Institutional Review Board at Shanghai Six People's Hospital, Shanghai Jiaotong University. UCB was overlaid onto separation medium and centrifuged following the protocols. The mononuclear cells were placed into plates pre-coated with type I rat tail collagen and cultured in EGM-2MV (Lonza, Walkersville, MD, USA). After 48 hours, non-adherent cells were removed. The medium was changed daily for 7 days and then every other day until the first passage. Early-passage EPCs (p2–6) were used in the subsequent experiments. For immunocytochemistry, briefly, the cells were fixed and then incubated with primary antibodies overnight, followed by incubation with secondary antibodies for one hour. Isotype irrelevant antibodies were used as negative controls. Nuclei were stained with DAPI (Invitrogen, Grand Island, NY, USA) for 5 min. The cells were then washed three times and viewed using a fluorescence microscope (Leica AF6000). The antibodies, including mouse anti-human CD31 (1:100), rabbit anti-human CD34 (1:100), rabbit anti-human vWF(1:100), were obtained from Abcam (Cambridge, Cambs, UK). Fluorescent staining was used to detect the uptake of Dil-ac-LDL (Molecular Probes, Eugene, OR, USA) and binding of FITC-UEA-l (Sigma-Aldrich, St. Louis, MO). Briefly, the cells were incubated with Dil-ac-LDL (15 μg/mL) for 4 hours, and then stained with FITC-UEA-l (10 μg/mL) for one hour and with DAPI for 5 min. The cells were washed three times and analyzed under a fluorescence microscope (Leica AF6000). For flow cytometry, cells were fixed and then incubated with the following primary antibodies: FITC-conjugated mouse anti-human CD31, APC-conjugated mouse anti-human CD34, PE-conjugated mouse anti-human VEGFR-2, and FITC-conjugated mouse anti-human CD45. The antibodies were obtained from BD Biosciences (San Jose, CA, USA). Non-specific fluorescence was determined by incubation of similar cell aliquots with isotype-matched mouse monoclonal antibodies. The cells were washed with PBS and analyzed by using the Guava easyCyte™ Flow Cytometer (Millipore, Billerica, MA, USA). 2.2. Isolation and identification of exosomes released from human UCBderived EPCs The methods were performed as described previously (Li et al., 2016). Briefly, 80% confluent EPCs cultured for an additional 24 hours in EGM-2MV media deprived of FBS and supplemented with 1 × serum replacement solution (PeproTech, Rocky Hill, NJ, USA). The conditioned media of EPCs was obtained and centrifuged at 300 × g for 10 min and 2000 × g for 10 min at 4 °C to remove dead cells and cellular debris. Subsequently, the supernatant was filtered using a 0.22 μm filter sterilize Steritop TM (Millipore) to remove residual dead cells and cellular debris. Hereafter, the supernatant was centrifuged at 4000 × g at 4 °C to about 200 μL by ultra filtration in a
15 mL Amicon Ultra-15 Centrifugal Filter Unit (Millipore) and then centrifuged at 100,000 ×g for one hour at 4 °C to pellet the small vesicles. Exosomes were stored at − 80 °C or used for downstream experiments. The protein concentration of the exosomes was determined using the Micro Bicinchoninic Acid (BCA) Protein Assay Kit (Thermo Fisher, Waltham, MA, USA). Exosomes were visualized using a Hitachi H-7650 transmission electron microscope (Hitachi, Tokyo, Japan). Western blots were used to analyze the incorporation of each protein into exosomes. The antibodies against CD9 (1:300), CD63 (1:300) and CD81 (1:300), were obtained from Abcam. 2.3. Establishment of diabetic rats wound model and treatment All procedures were approved by the Animal Research Committee of the Sixth People's Hospital at the Shanghai Jiao Tong University. Male SD rats (250–300 g) were intravenously injected at a dose of 45 mg/kg of STZ (Sigma-Aldrich, St.Louis, USA). After four weeks, rats with fasting blood glucose levels higher than 16.67 mmol/L were considered diabetic and included for the experiments. Rats were anesthetized by intraperitoneal injection of 75 mg/kg pentobarbital, and after shaving a full thickness of the excision wound of 2.0 cm in length and 0.2 cm depth was created. Rats were randomly assigned to 2 different treatment groups, which were subcutaneously injected at wound sites with 100 μL PBS (control group) or EPC-Exos (100 μg) around the wounds at four sites. 2.4. Histological analysis The wound skin and surrounding skin were perfused for with heparinized saline and fixed by formalin. Five sections (5-μm thickness) were collected on glass microscopy slides. Hematoxylin and eosin (H&E) staining was performed according to standard protocols. Scar width (mm) meaning the junction gap between the normal dermis and dermis in the wound tissues was measured. A LEICA DM 4000B microscope and LEICA application suite 3.8 software were used to obtain and analyze images. 2.5. In vitro effects of EPC-Exos on vascular endothelial cells Vascular endothelial cells from the human microvascular endothelial cell line, HMEC-1 (Center for Disease Control and Prevention, Atlanta, GA) were cultured in MCDB131 cell culture media (Gibco BRL, Grand Island, NY, USA) supplemented with 10% FBS (Gibco BRL). A Cell Counting Kit-8 assay (CCK-8; Dojindo, Kyushu Island, Japan) was used to assess cell proliferation. HMECs were seeded onto 96-well plates with exosomes (100 μg/mL) or PBS. At day 1, 2, 3, 4 and 5, CCK-8 solution (10 μL/well) was added to the HMECs. The absorbance was measured at 450 nm using a microplate reader. The optical density (OD) values represented the survival/proliferation of HMECs. The migration effects of exosomes on cells were evaluated in scratch assays. HMECs were plated in plates, and the confluent monolayer was scratched using a p200 pipette tip (1 mL) of serum-free MCDB131 media supplemented with exosomes (100 μg/mL) or PBS was added. The cells were photographed after 4 hours (t = 4 hours) and 8 hours (t = 8 hours). Wound areas were measured using the Image-Pro Plus 6.0 software. The capillary-network formation was monitored by performing tube-formation assays. HMECs were seeded onto Matrigel-coated 96-well plates and incubated in serum-free MCDB131 media supplemented with exosomes (100 μg/mL) or an equal volume of PBS. The cells were incubated for 4 hours (t = 4 hours) and 8 hours (t = 8 hours). The number of the total branch points and tubule lengths in 5 randomly chosen fields were examined using an inverted microscope. EPCs were labeled with Vybrant DiO dye (Molecular Probes, Carlsbad, CA, USA), according to the manufacturer's protocol. Briefly,
Please cite this article as: Li, X., et al., Human endothelial progenitor cells-derived exosomes accelerate cutaneous wound healing in diabetic rats by promoting endothelial function, Journal of Diabetes and Its Complications (2016), http://dx.doi.org/10.1016/j.jdiacomp.2016.05.009
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the cells were labeled for 15 min. The cell-labeled suspension was centrifuged and the supernatant was discarded. The cells cultured for an additional 24 hours in serum-free EGM-2 MV. Subsequently, the exosomes were isolated from the culture medium, and incubated with HMECs for 2 hours. The cells were then washed and analyzed under a fluorescence microscope (Leica AF6000). Influence of exosomes on the expression of angiogenesis-related genes in endothelial cells was evaluated with qRT-PCR. HMECs were seeded in plates and cultured in serum-free MCDB131 media containing exosomes (100 μg/mL) or PBS for 24 hours. qRT-PCR analysis was performed using an ABI PRISM®7900HT System with SYBR Premix ExTaq™ II (Takara Biotechnology, Japan). GAPDH was employed as the housekeeping gene for internal normalization.
2.6. Statistical analysis All of the experiments were performed at least three times. The data are shown as the means ± SEM. Differences were analyzed using an unpaired Student's t-test. P values b 0.05 were considered statistically significant.
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3. Results 3.1. Identification of human UCB-derived EPCs and EPC-Exos Immunostaining and flow cytometry analysis were used to detect the expressions of cell-specific antigens in the EPCs colonies. The results indicated that EPCs showed the typical endothelial-like cobblestone morphology and expressed endothelial lineage markers, such as CD31, vWF, and VEGFR-2, and were negative for the hematopoietic cell-specific marker CD45. Moreover, these cells also expressed CD34 (Fig. 1A–C), which showed their stem cells identity. Additionally, these cells were further tested by incorporating ac-LDL and binding endothelial-specific lectin UEA-1 (Fig. 1D), which are characteristic features of endothelial lineage cells. These studies confirmed that EPCs had been successfully isolated from the human UCB. In transmission electron microscopy experiments, we found that the majority of exosomes exhibited a round-shaped morphology, with a size ranging from 40 to 80 nm (Fig. 1E). And Western blotting analysis indicated these exosomes expressed characteristic exosomal surface marker proteins: CD9, CD63 and CD81 (Fig. 1F).
Fig. 1. Identification of human UCB-derived EPCs and EPC-Exos. (A) The UCB-derived adherent cells showed the typical endothelial-like cobblestone morphology. Scale bar: 100 μm. (B) Immunostaining revealed that the adherent cells were positive for CD31, vWF and CD34. Nuclei were counterstained by DAPI. Scale bar: 50 μm. (C) Flow cytometric analysis showed that the EPCs-like cells were positive for CD31, CD34, and VEGFR-2, but negative for the hematopoietic cell-specific marker CD45, indicating their EPCs identity. (D) The UCB-derived EPCs were further defined by their ability to bind FITC-UEA-l and uptake Dil-ac-LDL. Scale bar: 50 μm. (E) Morphology of EPC-Exos under a transmission electron microscopy. Scale bar: 50 nm. (F) Western blotting analysis of exosomal surface marker proteins (including CD9, CD63, and CD81) in EPC-Exos.
Please cite this article as: Li, X., et al., Human endothelial progenitor cells-derived exosomes accelerate cutaneous wound healing in diabetic rats by promoting endothelial function, Journal of Diabetes and Its Complications (2016), http://dx.doi.org/10.1016/j.jdiacomp.2016.05.009
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Fig. 2. EPC-Exos promote wound healing in diabetic rats. (A) Gross view and the effects of treatment with PBS or EPC-Exos on wound closure at 7 and 14 days. *P b 0.05, and EPC-Exos vs. control (n = 8 in each group). (B) H&E staining of wound sections following treatment with PBS or EPC-Exos at 14 days post-wounding. The double-headed arrows indicate the edges of the scar. *P b 0.05, and EPC-Exos vs. control (n = 8 in each group).
3.2. EPC-Exos promote wound healing in diabetic rats In the animal model of type 1 diabetes, we evaluated wound healing in 2 groups of rats that were treated with PBS (control group) or EPC-Exos around the wound sites. As shown in Fig. 2A, rats treated with EPC-Exos showed accelerated wound closure than the control groups at days 7 and 14. (day 7: 68.46 ± 6.3% vs. 24.35 ± 5.6%; day 14: 94.22 ± 5.1% vs. 75.56 ± 7.4%; n = 8; P b 0.05). In addition, the scar lengths were markedly reduced in the EPC-Exos group, compared to the control groups. These data suggested that EPC-Exos could use for the treatment of diabetic wound. 3.3. Internalized EPC-Exos functional assays and angiogenesis-related genes expression of vascular endothelial cells Furthermore, we investigated the function of exosomes for endothelial cells by using a series of in vitro assays. The effect of
EPC-Exos on the proliferation of vascular endothelial cells was determined by CCK-8 cell counting analysis, indicating that the exosomes treatment markedly enhanced the proliferation of HMECs (Fig. 3A). A wound healing assay was used to exam the effect of EPC-Exos on the migration of endothelial cells, showing that these exosomes significantly enhanced the motility of HMECs (Fig. 3B). The branch numbers and tube length were measured to assess the ability of HMECs to form tubules. Compared with the control group, all indicators that evaluated the tube formation capability were profoundly increased in HMECs stimulated with exosomes (Fig. 3C). Taken together, our in vitro experiments indicated that EPC-Exos could activate an angiogenic program in vascular endothelial cells. Fluorescence microscopy analysis showed the DiO-labeled EPC-Exos were taken up and transferred to the perinuclear region of endothelial cells (Fig. 3D), indicating that the EPC-Exos entered into the target cells, or that exosomes were internalized by vascular endothelial cells.
Please cite this article as: Li, X., et al., Human endothelial progenitor cells-derived exosomes accelerate cutaneous wound healing in diabetic rats by promoting endothelial function, Journal of Diabetes and Its Complications (2016), http://dx.doi.org/10.1016/j.jdiacomp.2016.05.009
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Fig. 3. Internalized EPC-Exos functional assays and angiogenesis-related genes expression of vascular endothelial cells. (A) EPC-Exos promoted human microvascular endothelial cells (HMECs) proliferation as analyzed by Cell Counting Kit-8 assay. *P b 0.05. (B) EPC-Exos stimulated the migration of HMECs. Scale bar: 250 μm. (C) The in vitro angiogenesis assay showed that EPC-Exos enhanced the tube formation of HMECs. Scale bar: 100 μm. (D) Fluorescent microscopy analysis of DiO-labeled EPC-Exos internalization in HMECs. The green-labeled exosomes were visible in the perinuclear region of HMECs. Scale bar: 50 μm. (E) qRT-PCR analysis of the expression levels of angiogenesis-related genes in HMECs stimulated with EPC-Exos or an equal volume of PBS buffer. *P b 0.05.
Please cite this article as: Li, X., et al., Human endothelial progenitor cells-derived exosomes accelerate cutaneous wound healing in diabetic rats by promoting endothelial function, Journal of Diabetes and Its Complications (2016), http://dx.doi.org/10.1016/j.jdiacomp.2016.05.009
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To test whether angiogenesis-related genes expression was altered by internalization of EPC-Exos, qRT-PCR analysis was performed. As shown in Fig. 3E, multiple critical pro-angiogenic genes were profoundly up-regulated by more than 4-fold after treatment with EPC-Exos, including endothelial fibroblast growth factor1 (FGF-1), nitric oxide synthase (eNOS), interleukin-8 (IL-8), angiopoietin-1 (ANG-1), E-selectin, vascular endothelial growth factor A (VEGFA), vascular endothelial growth factor receptor 2 (VEGFR-2), and chemokine ligand-16 (CXCL-16) showed notable up-regulation in endothelial cells stimulated with exosomes. In addition, exosomes-stimulated endothelial cells showed significant down-regulation of matrix metalloprotein-9 (MMP-9). These data indicated that EPC-Exos modulated the endothelial cells partly through the expressions of various angiogenesis-related genes. 4. Discussions Herein, we revealed that EPCs-derived exosomes transplantation could accelerate cutaneous wound healing in diabetic rats. We also showed that EPC-Exos enhanced the proliferation, migration and tube formation of vascular endothelial cells in vitro. Furthermore, we found that endothelial cells stimulated with these exosomes would increase expression of angiogenesis-related molecules. Therefore, our findings indicated that EPC-Exos facilitate wound healing by positively modulating vascular endothelial cells function. Recent studies have suggested that EPCs enhance endothelial cells regeneration by paracrine mechanism, but not by differentiation into mature endothelial cells (Baker et al., 2013; Kim et al., 2010). Exosomes are key components of paracrine secretion, and represent an important cell-cell communication by delivery of functional RNAs and proteins (Al-Nedawi, Meehan, & Rak, 2009; Zhu & Fan, 2011). Previous studies reported that human EPCs-derived exosomes do not express human leukocyte antigen class I and class II antigens (Cantaluppi et al., 2012), therefore, there are not overt immune reactions in our study. The further application of exosomes is promising without the potential risks of stem cell therapy, such as maldifferentiation and tumorigenesis. Actually, exosome therapeutic tools have been demonstrated in various disease models, such as reducing cardiomyocytes hypertrophy and apoptosis (Gu et al., 2014), enhancing neoangiogenesis of human pancreatic islets (Cantaluppi et al., 2012), and improving neovascularization in a hindlimb ischemia model (Ranghino et al., 2012). Exosomes derived from human induced pluripotent stem cell-derived mesenchymal stem cells could accelerate cutaneous wound healing, reduce scar widths, and enhance the proliferation and migration of human umbilical vein endothelial cells and human dermal fibroblasts in vitro (Zhang et al., 2015). Exosomes derived from human urine derived stem cells could promote endothelial cells' proliferation, and tube formation, and accelerate wound closure and re-epithelization in rats (Yuan, Guan, Zhang, Zhang, & Li, 2016). However, few investigations have aimed to discuss exosomes derived from EPCs in wound healing. Comparing with the low EPCs number in peripheral blood of diabetic patients, EPCs derived from UCB are easily obtained and present a great proliferative potential (Ingram et al., 2004). Therefore, in present study, we derived the EPCs from human UCB and confirmed that cells showed typical morphology and specific surface antigens. The transmission electron microscopy revealed the majority of exosomes were 60–80 nm in diameter and expressed specific exosomal markers, such as CD9, CD63 and CD81, which was consistent with previous reports (Ribeiro, Zhu, Millard, & Fan, 2013). As we known, exosome small size protects them from phagocytosis by the mononuclear cell system, and the membrane structure assists in easily passing their components across the recipient cell membrane. Therefore, exosomes are more convenient to bring and transfer proteins and mRNAs/microRNAs into target cells and consequently change target cells (Ribeiro et al., 2013). Our data
revealed that the EPCs-secreted exosomes up-regulated the expression of pro-angiogenesis molecules in vascular endothelial cells, including FGF-1, VEGFA, VEGFR-2, ANG-1, E-selectin, CXCL-16, eNOS and IL-8. These pro-angiogenesis molecules are critical for regulation of blood vessel development, by promoting proliferation, migration, and tube formation on endothelial cells (Wong & Crawford, 2013). However, the mRNA levels of MMP-9 were remarkably decreased in endothelial cells stimulated with EPCs-derived exosomes. MMP-9 seems to be implicated in increased inflammation because they are expressed mainly by neutrophils and macrophages, which are both important to the inflammatory response. Studies suggest that the high levels of MMP-9 predict poor wound healing (Liu et al., 2009), and the delivery of MMP-9-siRNA could reduce MMP-9 expression in skin fibroblast cells to accelerate wound healing (Li et al., 2014). 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Please cite this article as: Li, X., et al., Human endothelial progenitor cells-derived exosomes accelerate cutaneous wound healing in diabetic rats by promoting endothelial function, Journal of Diabetes and Its Complications (2016), http://dx.doi.org/10.1016/j.jdiacomp.2016.05.009