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serum deprivation-induced osteocyte apoptosis and osteocyte-mediated osteoclastogenesis in vitro
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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Adipose mesenchymal stem cell-derived exosomes ameliorate hypoxia/serum deprivation-induced osteocyte apoptosis and osteocyte-mediated osteoclastogenesis in vitro Lin Ren a, b, Zi-jun Song c, Qing-wei Cai a, b, Rui-xin Chen a, b, Yang Zou a, b, Qiang Fu a, b, **, Yuan-yuan Ma a, b, * a b c
Department of Prosthodontics, Guanghua School of Stomatology, Hospital of Stomatology, Sun Yat-sen University, Guangzhou, 510055, PR China Guangdong Provincial Key Laboratory of Stomatology, Guangzhou, 510055, PR China Department of Periodontics, Hospital of Stomatology, Kunming Medical University, Kunming, 650031, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 November 2018 Accepted 16 November 2018 Available online xxx
Age-related skeletal changes is closely associated with imbalanced bone remodeling characterized by elevated osteocyte apoptosis and osteoclast activation. Since osteocytes are the commander of bone remodeling, attenuating increased osteocyte apoptosis may improve age-related bone loss. Exosomes, derived from mesenchymal stem cells, hold promising potential for cell-free therapy due to multiple abilities, such as promoting proliferation and suppressing apoptosis. We aimed to explore the effect of exosomes derived from adipose mesenchymal stem cell (ADSCs-exo) on osteocyte apoptosis and osteocyte-mediated osteoclastogenesis in vitro. The osteocyte-like cell line MLO-Y4 was used as a model, and apoptosis was induced by hypoxia and serum deprivation (H/SD). Our results showed that ADSCsexo noticeably reduced H/SD-induced apoptosis in MLO-Y4 cells via upregulating the radio of Bcl-2/ Bax, diminishing the production of reactive oxygen species and cytochrome c, and subsequent activation of caspase-9 and caspase-3. Additionally, ADSCs-exo lowered the expression of RANKL both at the mRNA and protein levels, as well as the ratio of RANKL/OPG at the gene level. As determined by tartrateresistant acid phosphatase staining, reduced osteoclastogenesis was further validated in bone marrow monocytes cultured under conditioned medium from exosome-treated MLO-Y4. Together, ADSCs-exo could antagonize H/SD induced osteocyte apoptosis and osteocyte-mediated osteoclastogenesis, indicating the therapeutic potential of ADSCs-exo in age-related bone disease. © 2018 Elsevier Inc. All rights reserved.
Keywords: Adipose-derived mesenchymal stem cells Exosomes Osteocyte Apoptosis Osteoclastogenesis
1. Introduction Advancing age causes age-related skeletal diseases, such as osteoporosis. The key issue related to bone loss is thought to be the imbalance of bone remodeling [1]. Osteocytes, the most numerous and long-lived bone cells buried within the bone matrix, are well
* Corresponding author. Guanghua School of Stomatology, Hospital of Stomatology, Sun Yat-sen University, No. 56 Lingyuanxi Road, Guangzhou, 510055, PR China. ** Corresponding author. Guanghua School of Stomatology, Hospital of Stomatology, Sun Yat-sen University, No. 56 Lingyuanxi Road, Guangzhou, 510055, PR China. E-mail addresses: [email protected] (L. Ren), [email protected] (Z.-j. Song), [email protected] (Q.-w. Cai), [email protected] (R.-x. Chen), [email protected] (Y. Zou), [email protected] (Q. Fu), [email protected] (Y.-y. Ma).
known for their orchestration of bone formation and bone resorption. A host of studies have illustrated skeletal aging is closely associated with increased osteocyte apoptosis [2,3]. Other studies indicate that osteoclasts accumulate around apoptotic osteocytes, supporting apoptotic or dying osteocytes acting as a beacon for osteoclast recruitment [4]. Specifically, elevated osteocyte apoptosis is accompanied by the rise in the receptor activator of the nuclear factor kappa-B ligand (RANKL), as well as the decline in osteoprotegerin (OPG) [5,6]. RANKL interacts with its receptor RANK, which is highly expressed by osteoclasts or their precursors, and is essential for osteoclast activation. In contrast, OPG is the decoy receptor of RANKL that binds to RANKL; therefore, osteoclastogenesis is inhibited. Considering the key action of osteocyte apoptosis in regulating bone resorption, blocking excessive apoptosis of osteocytes may ameliorate age-related bone disease. Increased osteocyte apoptosis during age may occur when cells
https://doi.org/10.1016/j.bbrc.2018.11.109 0006-291X/© 2018 Elsevier Inc. All rights reserved.
Please cite this article as: L. Ren et al., Adipose mesenchymal stem cell-derived exosomes ameliorate hypoxia/serum deprivation-induced osteocyte apoptosis and osteocyte-mediated osteoclastogenesis in vitro, Biochemical and Biophysical Research Communications, https:// doi.org/10.1016/j.bbrc.2018.11.109
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suffer from pathological metabolic processes such as the rise of oxidative stress, lack of hormones, and disruption of blood flow [7,8]. The stimulation of hypoxia and ischemia may bring about the excess generation of reactive oxygen species (ROS) and trigger apoptosis or death by means of several apoptotic signaling pathways, especially the mitochondria-dependent apoptotic pathways. ROS may act as a factor initiating cellular apoptosis through a mechanism of mitochondrial pathways, which occurs with a large release of cytochrome c, activated caspase-9 and caspase-3 [9]. These mitochondria-dependent apoptotic events are mediated by the Bcl-2 family of proteins that decide the fate of cells via both anti-apoptosis (Bcl-2, Bcl-X, Bcl-w, etc.) and pro-apoptosis (Bax, Bad, Bak, etc.) members [10]. Emerging evidence has confirmed the vital effects of mesenchymal stem cells (MSCs) paracrine, especially secreted exosomes, for tissue regeneration [11e13]. Transportation of induced pluripotent stem cell-derived exosomes can prevent cardiomyocyte apoptosis against rat ischemic myocardium [14]. In an in vitro model of Huntington's disease, exosomes from human ADSCs protect against mitochondrial dysfunction and cell apoptosis via downregulation of p53, Bax and cleaved caspase-3 [15]. Actually, exosomes are membrane-enclosed nanovesicles with a diameter of 30e150 nm, which can directly fuse with recipient cells and transport proteins and nucleic acids into these cells, thereby altering the activity of target cells. Among all types of MSCs for exosome therapies, adipose-derived mesenchymal stem cells (ADSCs) are more beneficial owing to abundance, low immunogenicity and minimal ethical consideration [16]. In the current study, the osteocyte-like cell line MLO-Y4 was adopted, and apoptosis was induced by hypoxia and serum deprivation (H/SD) to mimic osteocyte apoptosis under hypoxia and ischemia conditions during aging. We assessed whether ADSCsderived exosomes (ADSCs-exo) could alleviate H/SD induced apoptosis and osteocyte-mediated osteoclastogenesis. The results of the study may facilitate the development of exosome applications in age-related bone diseases.
supplemented with 20% FBS for 36 h. Next, bone marrow monocytes (non-adherent cells) were collected, and 4 106 cells/well were seeded on the 24-well plate with 20% FBS and 30 ng/ml M-CSF (R＆D system,USA) at 37 C in a humidified 5% CO2 atmosphere for 3 days. 2.2. Isolation and characterization of ADSCs-exo When 90% confluence was reached by ADSCs at passage 3e4, the culture medium was replaced with DMEM/F12 medium without FBS for 48 h. The conditioned medium was harvested from ASDCs. Dead cells and cell debris were discarded by sequential centrifuging at 300 g for 10 min, 2000 g for 20 min and 10000 g for 40 min at 4 C. The exosome pellets were further collected by ultracentrifugation at 110000 g for 70 min at 4 C. The isolated exosomes were washed with PBS and again underwent ultracentrifugation at 110000 g for 70 min. Final exosomes were resuspended in serum-free a-MEM for immediate use or stored at 80 C. Purified exosomes were identified by transmission electron microscopy (FEI TECNAI, USA). The size distribution was analyzed by Nanosizer TM technology (Nanosight NS300, Malvern, UK). The concentration of exosomes was detected according to a BCA Protein Assay Kit (Cowin, China). In addition, exosome markers CD9, CD63, and HSP70 were detected by western blot. 2.3. Exosome labeling and uptake ADSCs-exo were labeled using PKH67 Green Fluorescent Cell Linker Kits according to the manufacturer's instructions (Sigma, USA). Excess dye was removed by ultracentrifugation at 4 C at 110000 g for 70 min, twice using a 32Ti rotor (Beckman Coulter, USA). The labeled exosomes were incubated with the MLO-Y4 cells for 6 h and 12 h. After that, cells were fixed and stained with Rhodamine phalloidin (Invitrogen, USA) and 4,'6-diamidino-2phenylindole (Beyotime, China). The untaken of labeled exosomes by MLO-Y4 cells was obtained using a LSM780 confocal microscope (Zeiss, Germany).
2. Materials and methods 2.4. Measurement of cell viability and apoptosis 2.1. Cell culture Adipose tissue was harvested from the inguinal fat pads of C57 BL/6 mice aged 3e4 weeks. The tissue was mechanically chopped into pieces approximately 1 mm3 and digested with collagenase I (Sigma, USA) for 40 min at 37 C. After collection by centrifugation at 1000 rpm for 5 min, the cell pellet was resuspended in DMEM/ F12 supplemented with 10% fetal bovine serum (FBS) and cultured in a humidified 5% CO2 atmosphere at 37 C. The murine long bone osteocyte Y4 (MLO-Y4) cell line (kindly provided by Dr. Bonewald) was plated on type I collagen-coated dishes (BD Biosciences, USA) and was cultured in a-MEM supplemented with 2.5% FBS and 2.5% bovine calf serum at 37 C in a humidified 5% CO2 atmosphere. For the apoptosis treatment, cells were exposed in the presence or absence of serum medium under normoxia (20% O2) or hypoxia (1% O2) for 24 h. Then, 20 mg/ml of exosomes was added to the cell medium under H/SD conditions to examine the effect of ADSCs-exo on H/SD induced apoptosis. In brief, MLO-Y4 cells were randomly divided into 5 groups: N/S (normoxia and serum conditions), N/SD (normoxia and serum deprivation conditions), H/S (hypoxia and serum conditions), H/ SD þ exo (hypoxia and ADSCs-exo added serum deprivation conditions), and the H/SD group (hypoxia and serum deprivation conditions). Bone marrow cells were harvested from C57BL/6 mice by flushing out the bone marrow and then culturing cells with DMEM
MLO-Y4 cells were seeded into 96-well plates (7 103/well) and treated as mentioned above. Cell viability was examined using the Cell Counting Kit-8 (Dojindo, Japan). Optical density was measured at 450 nm using the Tecan SUNRISE microplate reader (Tecan, Switzerland). For apoptosis analysis, the cells were seeded into a 60-mm culture dish (5 105/dish). Detection of cell apoptosis rates was performed by flow cytometry using the FITC Annexin V Apoptosis Detection Kit I (BD biosciences, USA). Cells stained with AV only or AV and PI were considered early-stage apoptosis and late-stage apoptosis, respectively. 2.5. Measurement of intracellular ROS MLO-4Y cells were seeded in 6-well plates (2 105) and treated as mentioned before. After 24 h treatments, 20,70dichlorodihydrofluorescein diacetate (DCFH-DA; Invitrogen, USA) was added to the cells for 20 min. The image was determined by an inverted fluorescence microscope (Carl Zeiss, Germany). Fluorescence intensity was measured using Image-Pro Plus software. 2.6. Osteoclast formation and staining To evaluate osteoclast formation, the condition medium of Bone marrow monocytes (BMMs) was changed to serum-supplemented DMEM with 50 ng/ml sRANKL (R&D Systems, USA) and 30 ng/ml
Please cite this article as: L. Ren et al., Adipose mesenchymal stem cell-derived exosomes ameliorate hypoxia/serum deprivation-induced osteocyte apoptosis and osteocyte-mediated osteoclastogenesis in vitro, Biochemical and Biophysical Research Communications, https:// doi.org/10.1016/j.bbrc.2018.11.109
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MCSF (R&D Systems, USA) for 2 days. On day 3, conditioned medium of MLO-Y4 cells from above groups was added to BMMs in a 1:1 ratio with 30 ng/ml MCSF and 50 ng/ml sRANKL in serumsupplemented DMEM. On day 6, mature multinucleated osteoclasts were detected by tartrate-resistant acid phosphatase (TRAP) staining according to an Acid Phosphatase Leukocyte kit (Sigma, USA). Osteoclasts were identified as TRAP positive cells containing at least three nuclei (TRAP þ MNCs). Five microscopic fields were randomly selected at 100X magnification to count TRAP positive cells. 2.7. RNA extraction and quantitative real-time PCR Total RNA was isolated using Trizol reagent (Invitrogen, USA). Next, the mRNAs were reverse transcribed into cDNA following the PrimeScript® One Step RT PCR kit (Takara, Japan). Subsequently, SYBR® Premix Ex Taq™ II (Takara, Japan) was used for qPCR on an LightCycler 480 Real-Time PCR System (Rocha, USA). b-actin was used as the basic control for normalization. Relative expression of mRNA was calculated using a 2-DDCt value method. Primer sequences were listed as follows: b-actin, forward 50 -CTTTTCCAGCCTTCCTTCTTG-30 , reverse 50 -TTGGCATAGAGGTCTTTACGG-3'; Bcl-2, forward 50 GACATGGCTGCCAGGACGT-30 , reverse 50 -CACCCCATCCCTGAAGAGTT3'; Bax, forward 50 -GC CTCCTCTCCTACTTCGGG-30 , reverse 50 GAAAAGACACAGTCCAAGGCAG-3'; RANKL, forward 50 -GAAACATCGGGAAGCGTACCTAC-30 and reverse 50 -GCTCCCTCCTTTCATCAGGTTAT-3'; and OPG, forward 50 -GACCAAAGTGAATGCCGAGAG-30 and reverse 50 -CGCTGCTTTCACAGAGGTCAA-3'.
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the track of exosomes, MLO-Y4 cells were cultured with PKH67 labeled ADSCs-exo for 6 h and 12 h and scanned with a laser scanning confocal microscope. After 6 h of incubation and green fluorescence signal, PKH67-labeled exosomes were detected in MLO-Y4 cells. Most of exosomes were localized in the cytoplasm of cells after 12 h (Fig. 1D). 3.2. Anti-apoptotic effect of ADSCs-exo on MLO-Y4 cells under H/SD conditions To investigate the effect of ADSCs-exo on MLO-Y4 cells, the cells were treated with H/SD together with ADSCs-exo for 24 h. First, as shown in Fig. 2A, H/SD significantly reduced cell viability compared with untreated cells (the N/S group), while ADSCs-exo reversed the decline of cell viability under H/SD conditions. Next, anti-apoptotic effects of ADSCs-exo on MLO-Y4 cells were further examined. As determined by flow cytometry, a significantly lower percentage of apoptotic cells were found in the H/SD þ Exo group compared to the H/SD group. Additionally, the difference of apoptosis rates was not significant between the H/SD þ exo group and the H/S group (Fig. 2B and C). However, neither cell viability nor cell apoptosis in the H/SD þ exo group returned to the same level as that of the N/S group (Fig. 2A and B). These observations suggest that the H/SD treatment induced MLO-Y4 cell apoptosis, and the apoptosis could be attenuated by ADSCs-exo. 3.3. ADSCs-exo exerted resistance to H/SD induced apoptosis via attenuating ROS production and mitochondria-dependent signal activation
2.8. Western blot analysis Proteins from exosome-rich pellets or MLO-Y4 cells were extracted with ice cold RIPA buffer (Cell Signaling Technology, USA) containing phenylmethanesulfonyl fluoride (Cell Signaling Technology, USA). Equal amounts of total proteins (50 mg) were fractionated by SDS-polyacrylamide gel electrophoresis on a 12% gel and were transferred to polyvinylidene difluoride membranes (Millipore, USA). The membranes were blocked with 5% defatted milk and incubated at room temperature for 1 h. After blocking, the membranes were incubated overnight at 4 C with primary antibodies against: CD9 (Abcam, USA), CD63 (Abcam, USA), HSP70 (Cell Signaling Technology, USA), caspase 3 (Cell Signaling Technology, USA), caspase 9 (Cell Signaling Technology, USA), cytochrome c (Cell Signaling Technology, USA), RANKL (Abcam, USA), and btubulin (Emarbio Science &Technology, China). The membranes were then incubated with 1:2000 of a secondary antibody conjugated with horseradish peroxidase (Cell Signaling Technology, USA) for 1 h at room temperature. Proteins were visualized by an enhanced chemiluminescence detection system (General Electric Company, USA). All data are expressed as the mean ± standard deviation. Statistical differences were calculated by one-way ANOVA, followed by the Bonferroni multiple comparison test. P value＜0.05 was considered significant.
Excessive reactive oxygen species (ROS) are reported to be produced under hypoxia/ischemia conditions and can cause cell apoptosis or death through classical apoptotic mechanisms [17]. Therefore, levels of ROS were examined. As determined by fluorescence intensities of DCFH, H/SD significantly promoted a 3.7fold rise in ROS generation in the MLO-Y4 cells, while ADSCs-exo significantly attenuated the increase in ROS compared with the H/SD group (Fig. 3A and B). To further elucidate the mechanisms of anti-apoptosis of ADSCs-exo, some key effectors related to mitochondria-dependent pathways were assessed. To sum up, under H/SD conditions, ADSCs-exo significantly promoted antiapoptotic Bcl-2 mRNA, suppressed the expression of proapoptotic Bax mRNA, the protein expression of cytochrome c, and the protein levels of cleaved caspase-9 and cleaved caspase-3 (Fig. 3CeF). These apoptosis-related factors in the H/SD þ Exo group almost returned to the same level as that in the HS group; however, there was still a significant difference between the H/ SD þ exo group and the N/S group (Fig. 3C, D, F). The above findings revealed that ADSCs-exo displayed cytoprotection as effectively as the serum against H/SD injury, which was at least partly mediated by suppressing the production of ROS and activation of mitochondria-dependent pathways. 3.4. ADSCs-exo alleviated enhanced osteocyte-mediated osteoclastogenesis in MLO-Y4 cells exposed to H/SD conditions
3. Results 3.1. ADSCs-exo: characterization and internalization into MLO-Y4 cells The typical ‘cup-shade’ nanovesicle exosomes were captured by transmission electron microscopy (Fig. 1A). Additionally, a nanoparticle tracking system revealed most ADSCs-exo were 50e150 nm in diameter (Fig. 1B). The exosome markers CD9, CD63, and HSP70 were then confirmed by western blot (Fig. 1C). To trace
Osteocytes, known as the main source of RANKL, participate in regulating osteoclastogenesis greatly through RANKL and OPG [18]. Therefore, expression of RANKL and OPG were evaluated. As shown in Fig. 4A, ADSCs-exo retained low expression of RANKL mRNA under H/SD conditions. Conversely, the OPG mRNA was enhanced in the H/SD-exo group compared with the H/SD group. A significant difference in the ratio of RANKL/OPG at the gene level was found between the H/SD-exo group and the H/SD group, suggesting that exosomes abolished H/SD induced changes in osteocytes.
Please cite this article as: L. Ren et al., Adipose mesenchymal stem cell-derived exosomes ameliorate hypoxia/serum deprivation-induced osteocyte apoptosis and osteocyte-mediated osteoclastogenesis in vitro, Biochemical and Biophysical Research Communications, https:// doi.org/10.1016/j.bbrc.2018.11.109
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Fig. 1. Characterization of ADSCs-exo and their internalization into MLO-Y4 cells. (A) Morphology of ADSCs-exo using transmission electron microscopy. Scale bar, 100 nm. (B) Size distribution and concentration of ADSCs-exo were measured by nanoparticle tracking analysis. (C) Western blotting of exosome markers CD9, CD63, and HSP70. (D) Internalization of exosomes into MLO-Y4 cells was detected after MLO-Y4 cells (red fluorescence for the cytoskeleton and blue fluorescence for the nucleus) were cocultured with PKH-67-labeled exosomes (green fluorescence) for 6 h and 12 h, Scale bar, 20 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig. 2. ADSCs-exo improved MLO-Y4 cell viability and ameliorated cell apoptosis under H/SD conditions. (A) Viability of cells was assessed by CCK-8. (B, C) Flow cytometry of cell apoptotic rate, bar plot graphs represent total apoptotic rate including early-phase apoptotic cells and late-phase apoptotic cells. Data are represented as the mean ± standard : deviation of three independent experiments. Analysis of variance; *P < 0.05 vs. N/S group; #P < 0.05 vs. H/SD group; P < 0.05 vs. H/S group.
Meanwhile, consistent with the mRNA results, the protein expression of RANKL showed similar trends (Fig. 4B). However, we were not able to precisely measure OPG protein levels since the
values obtained by ELISA were too low. These results implied that the effect of ADSCs-exo on osteocyte-mediated osteoclastogenesis was mainly attributed to the change in RANKL expression.
Please cite this article as: L. Ren et al., Adipose mesenchymal stem cell-derived exosomes ameliorate hypoxia/serum deprivation-induced osteocyte apoptosis and osteocyte-mediated osteoclastogenesis in vitro, Biochemical and Biophysical Research Communications, https:// doi.org/10.1016/j.bbrc.2018.11.109
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Fig. 3. ADSCs-exo attenuated H/SD induced ROS generation and mitochondrial pathway activation. (A, B) ROS generation was evaluated by fluorescence microscopy; fluorescence intensity was calculated and the fluorescence intensity of the N/S group was set as the control. (C) qRT-PCR analysis of Bcl-2 mRNA, Bax mRNA and the ratio of Bcl-2/Bax; data were normalized to b-actin. (DeF) Representative western blot analysis of cytochrome c, cleaved caspase-9, and cleaved caspase-3; densitometric analysis was used to quantify the levels of these proteins, b-tubulin served as a loading control. All values are represented as the mean ± S.D. of at least three independent experiments. Analysis of variance; *P < 0.05 vs. N/ : S group; #P < 0.05 vs. H/SD group; P < 0.05 vs. H/S group.
Additionally, conditioned media from the H/SD-exo group induced a lower number of osteoclast-like cells compared with conditioned media from the H/SD group (Fig. 4C). Together, ADSCs-exo treated MLO-Y4 cells show decreased osteoclastogenic cytokines and osteoclastogenic potential under H/SD conditions.
4. Discussion Growing advances have highlighted the value of MSCs-derived exosomes for repairing tissue injury under hypoxia and ischemia conditions [19,20]. Some studies have revealed ADSCs-exo dramatically ameliorate ischemia/reperfusion (I/R) induced myocardial necrosis in rat myocardial injury models [21]. Qiancheng Luo et al. have found that coculture of H9c2 cells with ADSCsexo, especially coculture with exosomes from miR-126overexpressing ADSCs, can decrease the apoptotic cells promoted by hypoxia treatment [22]. In fact, MSCs-derived exosomes account for tissue repair not only by antagonizing apoptosis but also by pro-
angiogenic, pro-survival and immunomodulatory effects [11,20,23]. Such exosomes are a promising substitute for cell therapies, which avert the safety risks concerned with direct stem cell transplantation and are less technical to store and deliver [24]. Given the superiority of exosomes, MLO-Y4 cells were cocultured with ADSCs-exo under H/SD conditions, and our data revealed that ADSCs-exo remarkably reduced the H/SD induced drop of viability and rise of apoptosis. Recent evidence in animal and clinical studies has indicated that oxidative stress and ROS are strongly linked to the pathogenesis of age-related bone loss [25,26]. Indeed, oxidative stress can activate the pre-osteoclasts and induces apoptosis of osteoblasts and osteocytes, thus favoring bone resorption. On the other hand, some in vitro research demonstrated that a large quantity of ROS is produced when cells undergo apoptosis or death under H/SD conditions [17,27]. Similarly, the results of the present study also suggested that a significant rise of ROS appeared in MLO-Y4 cells when they were treated with H/SD, in particular, ADSCs-exo
Please cite this article as: L. Ren et al., Adipose mesenchymal stem cell-derived exosomes ameliorate hypoxia/serum deprivation-induced osteocyte apoptosis and osteocyte-mediated osteoclastogenesis in vitro, Biochemical and Biophysical Research Communications, https:// doi.org/10.1016/j.bbrc.2018.11.109
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Fig. 4. ADSCs-exo reduced osteoclastogenic potential in H/SD treated MLO-Y4 cells. (A) RANKL and OPG mRNA levels were examined by qRT-PCR, and the values were normalized with b-actin; the ratio of RANKL/OPG was calculated by the expression values obtained by qRT-PCR. (B) Protein expression of RANKL was detected by western blot, and b-tubulin served as a control. (C) TRAP-staining of BMMs cultured with conditioned media from MLO-Y4 cells; osteoclasts were identified as TRAP positive cells containing three or more nuclei (TRAP þ MNCs). “N/S-CM”, “N/SD-CM”, “H/S-CM”, “H/SD þ exo-CM” and “H/SD-CM” represented BMMs cultured with conditioned media from N/S treated MLO-Y4 cells, N/SD treated MLO-Y4 cells, H/S treated MLO-Y4 cells, H/SD þ exo treated MLO-Y4 cells and H/SD treated MLO-Y4 cells, respectively. All results are presented as the mean ± S.D. of at least three independent experiments. Analysis of variance; *P < 0.05 vs. N/S group; #P < 0.05 vs. H/SD group.
obviously inhibited excessive production of ROS stimulated by H/ SD. Apart from ROS, activation of mitochondrial-dependent apoptotic pathways is also reported to be responsible for cell death under hypoxia and ischemia stimulation [9]. A remarkable decrease in anti-apoptotic protein Bcl-2 is present in MLO-Y4 cells under a hypoxic-ischemic environment, while pro-apoptotic protein Bax appears in a contrary trend [28]. Our results also displayed the same trend for Bcl-2, and Bax expression at mRNA levels in MLO-Y4 cells in H/SD conditions. More importantly, ADSCs-exo could significantly reduce H/SD-induced downregulation of Bcl-2 and repress H/SD-induced upregulation of Bax. Moreover, factors including ROS and the Bcl-2 family of proteins may serve as upstream stimuli, which activate the mitochondria-dependent
apoptotic factors. Our results demonstrated ADSCs-exo effectively alleviated the release of cytochrome c, and the activation of caspase 9 and caspase-3 under H/SD conditions, which was similar to previous findings that the combination of ADSCs and ADSCs-exo can protect cardiomyocytes against I/R injury concomitant with downregulation of mitochondrial cytochrome c, Bax, cleaved caspase 3 and cleaved PARP [29]. In addition to inducing apoptosis, the H/SD treatment affected the expression of RANKL/OPG as well. Data have shown that apoptotic osteocytes may exhibit enhancement of osteoclastogenesis by elevated RANKL expression [6,30]. Increased RANKL, as well as decreased OPG, is reported by Domazetovic in MLO-Y4 cells for 24 h serum deprivation. It has been confirmed that the RANKL/ OPG ratio plays a vital role in regulating bone formation and bone
Please cite this article as: L. Ren et al., Adipose mesenchymal stem cell-derived exosomes ameliorate hypoxia/serum deprivation-induced osteocyte apoptosis and osteocyte-mediated osteoclastogenesis in vitro, Biochemical and Biophysical Research Communications, https:// doi.org/10.1016/j.bbrc.2018.11.109
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resorption [18]. In the current study, MLO-Y4 cells displayed a significant increase in RANKL levels and decreased OPG levels when cultured under H/SD conditions for 24 h. Particularly, the elevation of RANKL/OPG at the mRNA level and RANKL expression at the protein level were obviously reverted by ADSCs-exo under H/SD conditions, suggesting that exosomes may affect MLO-Y4-triggered osteoclastogenesis by regulating RANKL expression and the RANKL/ OPG ratio. As expected, less formation of multinucleated osteoclasts was found when BMMs were cultured with the condition medium from exosome treated osteocytes, indicating that ADSCsexo efficiently diminish osteocyte-mediated osteoclastogenesis under H/SD conditions. In conclusion, our study demonstrated, for the first time, that ADSCs-exo could effectively inhibit H/SD induced osteocyte apoptosis and osteocyte-mediated osteoclastogenesis. The antiapoptotic effects of ADSCs-exo were attained through the upregulation of Bcl-2/Bax, the suppression of ROS, cytochrome c generation and caspase-3, caspase-9 activation. Meanwhile, the inhibition of osteocyte-mediated osteoclastogenesis was obtained mainly via downregulation of the expression of RANKL. Animals studies should be required to further confirm the therapeutic effect of ADSCs for age-related bone diseases. Conflicts of interest The authors declare that they have no conflicts of interest. Acknowledgements This work was supported by the National Natural Science Foundation of China (Grant No. 81570955). This work was supported by Guangzhou Science and Technology and Innovation Commission (Grant No. 201510010222). Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2018.11.109. References [1] K.M. Nicks, S. Amin, E.J. Atkinson, B.L. Riggs, L.J. Melton, S. Khosla, Relationship of age to bone microstructure independent of areal bone mineral density, J. Bone Miner Res. 27 (2012) 637e644. https://doi.org/10.1002/jbmr.1468. [2] R.L. Jilka, C.A. O'Brien, The role of osteocytes in age-related bone loss, J. Curr. Osteoporos. Rep. 14 (2016) 16e25. https://doi.org/10.1007/s11914-016-02970. [3] D.M. Joiner, R.J. Tayim, J.D. McElderry, M.D. Morris, S.A. Goldstein, Aged male rats regenerate cortical bone with reduced osteocyte density and reduced secretion of nitric oxide after mechanical stimulation, J. Calcif. Tissue Int. 94 (2014) 484e494. https://doi.org/10.1007/s00223-013-9832-5. [4] T. Nakashima, M. Hayashi, T. Fukunaga, K. Kurata, M. Oh-Hora, J.Q. Feng, L.F. Bonewald, T. Kodama, A. Wutz, E.F. Wagner, J.M. Penninger, H. Takayanagi, Evidence for osteocyte regulation of bone homeostasis through RANKL expression, J. Nat. Med. 17 (2011) 1231e1234. https://doi.org/10.1038/nm. 2452. [5] S.A. Al-Dujaili, E. Lau, H. Al-Dujaili, K. Tsang, A. Guenther, L. You, Apoptotic osteocytes regulate osteoclast precursor recruitment and differentiation in vitro, J. Cell Biochem. 112 (2011) 2412e2423. https://doi.org/10.1002/jcb. 23164. [6] H.M. Davis, R. Pacheco-Costa, E.G. Atkinson, L.R. Brun, A.R. Gortazar, J. Harris, M. Hiasa, S.A. Bolarinwa, T. Yoneda, M. Ivan, A. Bruzzaniti, T. Bellido, L.I. Plotkin, Disruption of the Cx43/miR21 pathway leads to osteocyte apoptosis and increased osteoclastogenesis with aging, J. Aging Cell. 16 (2017) 551e563. https://doi.org/10.1111/acel.12586. [7] K. Kobayashi, H. Nojiri, Y. Saita, D. Morikawa, Y. Ozawa, K. Watanabe, et al., Mitochondrial superoxide in osteocytes perturbs canalicular networks in the setting of age-related osteoporosis, J. Sci. Rep. 5 (2015) 9148. https://doi.org/ 10.1038/srep09148. [8] A.E. Tami, P. Nasser, O. Verborgt, M.B. Schaffler, M.L.K. Tate, The role of interstitial fluid flow in the remodeling response to fatigue loading, J. Bone Miner Res. 17 (2002) 2030e2037. https://doi.org/10.1359/jbmr.2002.17.11.
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Adipose mesenchymal stem cell-derived exosomes ameliorate hypoxia/serum deprivation-induced osteocyte apoptosis and osteocyte-mediated osteoclastogenesis in vitro Lin Ren a, b, Zi-jun Song c, Qing-wei Cai a, b, Rui-xin Chen a, b, Yang Zou a, b, Qiang Fu a, b, **, Yuan-yuan Ma a, b, * a b c
Department of Prosthodontics, Guanghua School of Stomatology, Hospital of Stomatology, Sun Yat-sen University, Guangzhou, 510055, PR China Guangdong Provincial Key Laboratory of Stomatology, Guangzhou, 510055, PR China Department of Periodontics, Hospital of Stomatology, Kunming Medical University, Kunming, 650031, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 November 2018 Accepted 16 November 2018 Available online xxx
Age-related skeletal changes is closely associated with imbalanced bone remodeling characterized by elevated osteocyte apoptosis and osteoclast activation. Since osteocytes are the commander of bone remodeling, attenuating increased osteocyte apoptosis may improve age-related bone loss. Exosomes, derived from mesenchymal stem cells, hold promising potential for cell-free therapy due to multiple abilities, such as promoting proliferation and suppressing apoptosis. We aimed to explore the effect of exosomes derived from adipose mesenchymal stem cell (ADSCs-exo) on osteocyte apoptosis and osteocyte-mediated osteoclastogenesis in vitro. The osteocyte-like cell line MLO-Y4 was used as a model, and apoptosis was induced by hypoxia and serum deprivation (H/SD). Our results showed that ADSCsexo noticeably reduced H/SD-induced apoptosis in MLO-Y4 cells via upregulating the radio of Bcl-2/ Bax, diminishing the production of reactive oxygen species and cytochrome c, and subsequent activation of caspase-9 and caspase-3. Additionally, ADSCs-exo lowered the expression of RANKL both at the mRNA and protein levels, as well as the ratio of RANKL/OPG at the gene level. As determined by tartrateresistant acid phosphatase staining, reduced osteoclastogenesis was further validated in bone marrow monocytes cultured under conditioned medium from exosome-treated MLO-Y4. Together, ADSCs-exo could antagonize H/SD induced osteocyte apoptosis and osteocyte-mediated osteoclastogenesis, indicating the therapeutic potential of ADSCs-exo in age-related bone disease. © 2018 Elsevier Inc. All rights reserved.
Keywords: Adipose-derived mesenchymal stem cells Exosomes Osteocyte Apoptosis Osteoclastogenesis
1. Introduction Advancing age causes age-related skeletal diseases, such as osteoporosis. The key issue related to bone loss is thought to be the imbalance of bone remodeling [1]. Osteocytes, the most numerous and long-lived bone cells buried within the bone matrix, are well
* Corresponding author. Guanghua School of Stomatology, Hospital of Stomatology, Sun Yat-sen University, No. 56 Lingyuanxi Road, Guangzhou, 510055, PR China. ** Corresponding author. Guanghua School of Stomatology, Hospital of Stomatology, Sun Yat-sen University, No. 56 Lingyuanxi Road, Guangzhou, 510055, PR China. E-mail addresses: [email protected] (L. Ren), [email protected] (Z.-j. Song), [email protected] (Q.-w. Cai), [email protected] (R.-x. Chen), [email protected] (Y. Zou), [email protected] (Q. Fu), [email protected] (Y.-y. Ma).
known for their orchestration of bone formation and bone resorption. A host of studies have illustrated skeletal aging is closely associated with increased osteocyte apoptosis [2,3]. Other studies indicate that osteoclasts accumulate around apoptotic osteocytes, supporting apoptotic or dying osteocytes acting as a beacon for osteoclast recruitment [4]. Specifically, elevated osteocyte apoptosis is accompanied by the rise in the receptor activator of the nuclear factor kappa-B ligand (RANKL), as well as the decline in osteoprotegerin (OPG) [5,6]. RANKL interacts with its receptor RANK, which is highly expressed by osteoclasts or their precursors, and is essential for osteoclast activation. In contrast, OPG is the decoy receptor of RANKL that binds to RANKL; therefore, osteoclastogenesis is inhibited. Considering the key action of osteocyte apoptosis in regulating bone resorption, blocking excessive apoptosis of osteocytes may ameliorate age-related bone disease. Increased osteocyte apoptosis during age may occur when cells
https://doi.org/10.1016/j.bbrc.2018.11.109 0006-291X/© 2018 Elsevier Inc. All rights reserved.
Please cite this article as: L. Ren et al., Adipose mesenchymal stem cell-derived exosomes ameliorate hypoxia/serum deprivation-induced osteocyte apoptosis and osteocyte-mediated osteoclastogenesis in vitro, Biochemical and Biophysical Research Communications, https:// doi.org/10.1016/j.bbrc.2018.11.109
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suffer from pathological metabolic processes such as the rise of oxidative stress, lack of hormones, and disruption of blood flow [7,8]. The stimulation of hypoxia and ischemia may bring about the excess generation of reactive oxygen species (ROS) and trigger apoptosis or death by means of several apoptotic signaling pathways, especially the mitochondria-dependent apoptotic pathways. ROS may act as a factor initiating cellular apoptosis through a mechanism of mitochondrial pathways, which occurs with a large release of cytochrome c, activated caspase-9 and caspase-3 [9]. These mitochondria-dependent apoptotic events are mediated by the Bcl-2 family of proteins that decide the fate of cells via both anti-apoptosis (Bcl-2, Bcl-X, Bcl-w, etc.) and pro-apoptosis (Bax, Bad, Bak, etc.) members [10]. Emerging evidence has confirmed the vital effects of mesenchymal stem cells (MSCs) paracrine, especially secreted exosomes, for tissue regeneration [11e13]. Transportation of induced pluripotent stem cell-derived exosomes can prevent cardiomyocyte apoptosis against rat ischemic myocardium [14]. In an in vitro model of Huntington's disease, exosomes from human ADSCs protect against mitochondrial dysfunction and cell apoptosis via downregulation of p53, Bax and cleaved caspase-3 [15]. Actually, exosomes are membrane-enclosed nanovesicles with a diameter of 30e150 nm, which can directly fuse with recipient cells and transport proteins and nucleic acids into these cells, thereby altering the activity of target cells. Among all types of MSCs for exosome therapies, adipose-derived mesenchymal stem cells (ADSCs) are more beneficial owing to abundance, low immunogenicity and minimal ethical consideration [16]. In the current study, the osteocyte-like cell line MLO-Y4 was adopted, and apoptosis was induced by hypoxia and serum deprivation (H/SD) to mimic osteocyte apoptosis under hypoxia and ischemia conditions during aging. We assessed whether ADSCsderived exosomes (ADSCs-exo) could alleviate H/SD induced apoptosis and osteocyte-mediated osteoclastogenesis. The results of the study may facilitate the development of exosome applications in age-related bone diseases.
supplemented with 20% FBS for 36 h. Next, bone marrow monocytes (non-adherent cells) were collected, and 4 106 cells/well were seeded on the 24-well plate with 20% FBS and 30 ng/ml M-CSF (R＆D system,USA) at 37 C in a humidified 5% CO2 atmosphere for 3 days. 2.2. Isolation and characterization of ADSCs-exo When 90% confluence was reached by ADSCs at passage 3e4, the culture medium was replaced with DMEM/F12 medium without FBS for 48 h. The conditioned medium was harvested from ASDCs. Dead cells and cell debris were discarded by sequential centrifuging at 300 g for 10 min, 2000 g for 20 min and 10000 g for 40 min at 4 C. The exosome pellets were further collected by ultracentrifugation at 110000 g for 70 min at 4 C. The isolated exosomes were washed with PBS and again underwent ultracentrifugation at 110000 g for 70 min. Final exosomes were resuspended in serum-free a-MEM for immediate use or stored at 80 C. Purified exosomes were identified by transmission electron microscopy (FEI TECNAI, USA). The size distribution was analyzed by Nanosizer TM technology (Nanosight NS300, Malvern, UK). The concentration of exosomes was detected according to a BCA Protein Assay Kit (Cowin, China). In addition, exosome markers CD9, CD63, and HSP70 were detected by western blot. 2.3. Exosome labeling and uptake ADSCs-exo were labeled using PKH67 Green Fluorescent Cell Linker Kits according to the manufacturer's instructions (Sigma, USA). Excess dye was removed by ultracentrifugation at 4 C at 110000 g for 70 min, twice using a 32Ti rotor (Beckman Coulter, USA). The labeled exosomes were incubated with the MLO-Y4 cells for 6 h and 12 h. After that, cells were fixed and stained with Rhodamine phalloidin (Invitrogen, USA) and 4,'6-diamidino-2phenylindole (Beyotime, China). The untaken of labeled exosomes by MLO-Y4 cells was obtained using a LSM780 confocal microscope (Zeiss, Germany).
2. Materials and methods 2.4. Measurement of cell viability and apoptosis 2.1. Cell culture Adipose tissue was harvested from the inguinal fat pads of C57 BL/6 mice aged 3e4 weeks. The tissue was mechanically chopped into pieces approximately 1 mm3 and digested with collagenase I (Sigma, USA) for 40 min at 37 C. After collection by centrifugation at 1000 rpm for 5 min, the cell pellet was resuspended in DMEM/ F12 supplemented with 10% fetal bovine serum (FBS) and cultured in a humidified 5% CO2 atmosphere at 37 C. The murine long bone osteocyte Y4 (MLO-Y4) cell line (kindly provided by Dr. Bonewald) was plated on type I collagen-coated dishes (BD Biosciences, USA) and was cultured in a-MEM supplemented with 2.5% FBS and 2.5% bovine calf serum at 37 C in a humidified 5% CO2 atmosphere. For the apoptosis treatment, cells were exposed in the presence or absence of serum medium under normoxia (20% O2) or hypoxia (1% O2) for 24 h. Then, 20 mg/ml of exosomes was added to the cell medium under H/SD conditions to examine the effect of ADSCs-exo on H/SD induced apoptosis. In brief, MLO-Y4 cells were randomly divided into 5 groups: N/S (normoxia and serum conditions), N/SD (normoxia and serum deprivation conditions), H/S (hypoxia and serum conditions), H/ SD þ exo (hypoxia and ADSCs-exo added serum deprivation conditions), and the H/SD group (hypoxia and serum deprivation conditions). Bone marrow cells were harvested from C57BL/6 mice by flushing out the bone marrow and then culturing cells with DMEM
MLO-Y4 cells were seeded into 96-well plates (7 103/well) and treated as mentioned above. Cell viability was examined using the Cell Counting Kit-8 (Dojindo, Japan). Optical density was measured at 450 nm using the Tecan SUNRISE microplate reader (Tecan, Switzerland). For apoptosis analysis, the cells were seeded into a 60-mm culture dish (5 105/dish). Detection of cell apoptosis rates was performed by flow cytometry using the FITC Annexin V Apoptosis Detection Kit I (BD biosciences, USA). Cells stained with AV only or AV and PI were considered early-stage apoptosis and late-stage apoptosis, respectively. 2.5. Measurement of intracellular ROS MLO-4Y cells were seeded in 6-well plates (2 105) and treated as mentioned before. After 24 h treatments, 20,70dichlorodihydrofluorescein diacetate (DCFH-DA; Invitrogen, USA) was added to the cells for 20 min. The image was determined by an inverted fluorescence microscope (Carl Zeiss, Germany). Fluorescence intensity was measured using Image-Pro Plus software. 2.6. Osteoclast formation and staining To evaluate osteoclast formation, the condition medium of Bone marrow monocytes (BMMs) was changed to serum-supplemented DMEM with 50 ng/ml sRANKL (R&D Systems, USA) and 30 ng/ml
Please cite this article as: L. Ren et al., Adipose mesenchymal stem cell-derived exosomes ameliorate hypoxia/serum deprivation-induced osteocyte apoptosis and osteocyte-mediated osteoclastogenesis in vitro, Biochemical and Biophysical Research Communications, https:// doi.org/10.1016/j.bbrc.2018.11.109
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MCSF (R&D Systems, USA) for 2 days. On day 3, conditioned medium of MLO-Y4 cells from above groups was added to BMMs in a 1:1 ratio with 30 ng/ml MCSF and 50 ng/ml sRANKL in serumsupplemented DMEM. On day 6, mature multinucleated osteoclasts were detected by tartrate-resistant acid phosphatase (TRAP) staining according to an Acid Phosphatase Leukocyte kit (Sigma, USA). Osteoclasts were identified as TRAP positive cells containing at least three nuclei (TRAP þ MNCs). Five microscopic fields were randomly selected at 100X magnification to count TRAP positive cells. 2.7. RNA extraction and quantitative real-time PCR Total RNA was isolated using Trizol reagent (Invitrogen, USA). Next, the mRNAs were reverse transcribed into cDNA following the PrimeScript® One Step RT PCR kit (Takara, Japan). Subsequently, SYBR® Premix Ex Taq™ II (Takara, Japan) was used for qPCR on an LightCycler 480 Real-Time PCR System (Rocha, USA). b-actin was used as the basic control for normalization. Relative expression of mRNA was calculated using a 2-DDCt value method. Primer sequences were listed as follows: b-actin, forward 50 -CTTTTCCAGCCTTCCTTCTTG-30 , reverse 50 -TTGGCATAGAGGTCTTTACGG-3'; Bcl-2, forward 50 GACATGGCTGCCAGGACGT-30 , reverse 50 -CACCCCATCCCTGAAGAGTT3'; Bax, forward 50 -GC CTCCTCTCCTACTTCGGG-30 , reverse 50 GAAAAGACACAGTCCAAGGCAG-3'; RANKL, forward 50 -GAAACATCGGGAAGCGTACCTAC-30 and reverse 50 -GCTCCCTCCTTTCATCAGGTTAT-3'; and OPG, forward 50 -GACCAAAGTGAATGCCGAGAG-30 and reverse 50 -CGCTGCTTTCACAGAGGTCAA-3'.
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the track of exosomes, MLO-Y4 cells were cultured with PKH67 labeled ADSCs-exo for 6 h and 12 h and scanned with a laser scanning confocal microscope. After 6 h of incubation and green fluorescence signal, PKH67-labeled exosomes were detected in MLO-Y4 cells. Most of exosomes were localized in the cytoplasm of cells after 12 h (Fig. 1D). 3.2. Anti-apoptotic effect of ADSCs-exo on MLO-Y4 cells under H/SD conditions To investigate the effect of ADSCs-exo on MLO-Y4 cells, the cells were treated with H/SD together with ADSCs-exo for 24 h. First, as shown in Fig. 2A, H/SD significantly reduced cell viability compared with untreated cells (the N/S group), while ADSCs-exo reversed the decline of cell viability under H/SD conditions. Next, anti-apoptotic effects of ADSCs-exo on MLO-Y4 cells were further examined. As determined by flow cytometry, a significantly lower percentage of apoptotic cells were found in the H/SD þ Exo group compared to the H/SD group. Additionally, the difference of apoptosis rates was not significant between the H/SD þ exo group and the H/S group (Fig. 2B and C). However, neither cell viability nor cell apoptosis in the H/SD þ exo group returned to the same level as that of the N/S group (Fig. 2A and B). These observations suggest that the H/SD treatment induced MLO-Y4 cell apoptosis, and the apoptosis could be attenuated by ADSCs-exo. 3.3. ADSCs-exo exerted resistance to H/SD induced apoptosis via attenuating ROS production and mitochondria-dependent signal activation
2.8. Western blot analysis Proteins from exosome-rich pellets or MLO-Y4 cells were extracted with ice cold RIPA buffer (Cell Signaling Technology, USA) containing phenylmethanesulfonyl fluoride (Cell Signaling Technology, USA). Equal amounts of total proteins (50 mg) were fractionated by SDS-polyacrylamide gel electrophoresis on a 12% gel and were transferred to polyvinylidene difluoride membranes (Millipore, USA). The membranes were blocked with 5% defatted milk and incubated at room temperature for 1 h. After blocking, the membranes were incubated overnight at 4 C with primary antibodies against: CD9 (Abcam, USA), CD63 (Abcam, USA), HSP70 (Cell Signaling Technology, USA), caspase 3 (Cell Signaling Technology, USA), caspase 9 (Cell Signaling Technology, USA), cytochrome c (Cell Signaling Technology, USA), RANKL (Abcam, USA), and btubulin (Emarbio Science &Technology, China). The membranes were then incubated with 1:2000 of a secondary antibody conjugated with horseradish peroxidase (Cell Signaling Technology, USA) for 1 h at room temperature. Proteins were visualized by an enhanced chemiluminescence detection system (General Electric Company, USA). All data are expressed as the mean ± standard deviation. Statistical differences were calculated by one-way ANOVA, followed by the Bonferroni multiple comparison test. P value＜0.05 was considered significant.
Excessive reactive oxygen species (ROS) are reported to be produced under hypoxia/ischemia conditions and can cause cell apoptosis or death through classical apoptotic mechanisms [17]. Therefore, levels of ROS were examined. As determined by fluorescence intensities of DCFH, H/SD significantly promoted a 3.7fold rise in ROS generation in the MLO-Y4 cells, while ADSCs-exo significantly attenuated the increase in ROS compared with the H/SD group (Fig. 3A and B). To further elucidate the mechanisms of anti-apoptosis of ADSCs-exo, some key effectors related to mitochondria-dependent pathways were assessed. To sum up, under H/SD conditions, ADSCs-exo significantly promoted antiapoptotic Bcl-2 mRNA, suppressed the expression of proapoptotic Bax mRNA, the protein expression of cytochrome c, and the protein levels of cleaved caspase-9 and cleaved caspase-3 (Fig. 3CeF). These apoptosis-related factors in the H/SD þ Exo group almost returned to the same level as that in the HS group; however, there was still a significant difference between the H/ SD þ exo group and the N/S group (Fig. 3C, D, F). The above findings revealed that ADSCs-exo displayed cytoprotection as effectively as the serum against H/SD injury, which was at least partly mediated by suppressing the production of ROS and activation of mitochondria-dependent pathways. 3.4. ADSCs-exo alleviated enhanced osteocyte-mediated osteoclastogenesis in MLO-Y4 cells exposed to H/SD conditions
3. Results 3.1. ADSCs-exo: characterization and internalization into MLO-Y4 cells The typical ‘cup-shade’ nanovesicle exosomes were captured by transmission electron microscopy (Fig. 1A). Additionally, a nanoparticle tracking system revealed most ADSCs-exo were 50e150 nm in diameter (Fig. 1B). The exosome markers CD9, CD63, and HSP70 were then confirmed by western blot (Fig. 1C). To trace
Osteocytes, known as the main source of RANKL, participate in regulating osteoclastogenesis greatly through RANKL and OPG [18]. Therefore, expression of RANKL and OPG were evaluated. As shown in Fig. 4A, ADSCs-exo retained low expression of RANKL mRNA under H/SD conditions. Conversely, the OPG mRNA was enhanced in the H/SD-exo group compared with the H/SD group. A significant difference in the ratio of RANKL/OPG at the gene level was found between the H/SD-exo group and the H/SD group, suggesting that exosomes abolished H/SD induced changes in osteocytes.
Please cite this article as: L. Ren et al., Adipose mesenchymal stem cell-derived exosomes ameliorate hypoxia/serum deprivation-induced osteocyte apoptosis and osteocyte-mediated osteoclastogenesis in vitro, Biochemical and Biophysical Research Communications, https:// doi.org/10.1016/j.bbrc.2018.11.109
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Fig. 1. Characterization of ADSCs-exo and their internalization into MLO-Y4 cells. (A) Morphology of ADSCs-exo using transmission electron microscopy. Scale bar, 100 nm. (B) Size distribution and concentration of ADSCs-exo were measured by nanoparticle tracking analysis. (C) Western blotting of exosome markers CD9, CD63, and HSP70. (D) Internalization of exosomes into MLO-Y4 cells was detected after MLO-Y4 cells (red fluorescence for the cytoskeleton and blue fluorescence for the nucleus) were cocultured with PKH-67-labeled exosomes (green fluorescence) for 6 h and 12 h, Scale bar, 20 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig. 2. ADSCs-exo improved MLO-Y4 cell viability and ameliorated cell apoptosis under H/SD conditions. (A) Viability of cells was assessed by CCK-8. (B, C) Flow cytometry of cell apoptotic rate, bar plot graphs represent total apoptotic rate including early-phase apoptotic cells and late-phase apoptotic cells. Data are represented as the mean ± standard : deviation of three independent experiments. Analysis of variance; *P < 0.05 vs. N/S group; #P < 0.05 vs. H/SD group; P < 0.05 vs. H/S group.
Meanwhile, consistent with the mRNA results, the protein expression of RANKL showed similar trends (Fig. 4B). However, we were not able to precisely measure OPG protein levels since the
values obtained by ELISA were too low. These results implied that the effect of ADSCs-exo on osteocyte-mediated osteoclastogenesis was mainly attributed to the change in RANKL expression.
Please cite this article as: L. Ren et al., Adipose mesenchymal stem cell-derived exosomes ameliorate hypoxia/serum deprivation-induced osteocyte apoptosis and osteocyte-mediated osteoclastogenesis in vitro, Biochemical and Biophysical Research Communications, https:// doi.org/10.1016/j.bbrc.2018.11.109
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Fig. 3. ADSCs-exo attenuated H/SD induced ROS generation and mitochondrial pathway activation. (A, B) ROS generation was evaluated by fluorescence microscopy; fluorescence intensity was calculated and the fluorescence intensity of the N/S group was set as the control. (C) qRT-PCR analysis of Bcl-2 mRNA, Bax mRNA and the ratio of Bcl-2/Bax; data were normalized to b-actin. (DeF) Representative western blot analysis of cytochrome c, cleaved caspase-9, and cleaved caspase-3; densitometric analysis was used to quantify the levels of these proteins, b-tubulin served as a loading control. All values are represented as the mean ± S.D. of at least three independent experiments. Analysis of variance; *P < 0.05 vs. N/ : S group; #P < 0.05 vs. H/SD group; P < 0.05 vs. H/S group.
Additionally, conditioned media from the H/SD-exo group induced a lower number of osteoclast-like cells compared with conditioned media from the H/SD group (Fig. 4C). Together, ADSCs-exo treated MLO-Y4 cells show decreased osteoclastogenic cytokines and osteoclastogenic potential under H/SD conditions.
4. Discussion Growing advances have highlighted the value of MSCs-derived exosomes for repairing tissue injury under hypoxia and ischemia conditions [19,20]. Some studies have revealed ADSCs-exo dramatically ameliorate ischemia/reperfusion (I/R) induced myocardial necrosis in rat myocardial injury models [21]. Qiancheng Luo et al. have found that coculture of H9c2 cells with ADSCsexo, especially coculture with exosomes from miR-126overexpressing ADSCs, can decrease the apoptotic cells promoted by hypoxia treatment [22]. In fact, MSCs-derived exosomes account for tissue repair not only by antagonizing apoptosis but also by pro-
angiogenic, pro-survival and immunomodulatory effects [11,20,23]. Such exosomes are a promising substitute for cell therapies, which avert the safety risks concerned with direct stem cell transplantation and are less technical to store and deliver [24]. Given the superiority of exosomes, MLO-Y4 cells were cocultured with ADSCs-exo under H/SD conditions, and our data revealed that ADSCs-exo remarkably reduced the H/SD induced drop of viability and rise of apoptosis. Recent evidence in animal and clinical studies has indicated that oxidative stress and ROS are strongly linked to the pathogenesis of age-related bone loss [25,26]. Indeed, oxidative stress can activate the pre-osteoclasts and induces apoptosis of osteoblasts and osteocytes, thus favoring bone resorption. On the other hand, some in vitro research demonstrated that a large quantity of ROS is produced when cells undergo apoptosis or death under H/SD conditions [17,27]. Similarly, the results of the present study also suggested that a significant rise of ROS appeared in MLO-Y4 cells when they were treated with H/SD, in particular, ADSCs-exo
Please cite this article as: L. Ren et al., Adipose mesenchymal stem cell-derived exosomes ameliorate hypoxia/serum deprivation-induced osteocyte apoptosis and osteocyte-mediated osteoclastogenesis in vitro, Biochemical and Biophysical Research Communications, https:// doi.org/10.1016/j.bbrc.2018.11.109
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Fig. 4. ADSCs-exo reduced osteoclastogenic potential in H/SD treated MLO-Y4 cells. (A) RANKL and OPG mRNA levels were examined by qRT-PCR, and the values were normalized with b-actin; the ratio of RANKL/OPG was calculated by the expression values obtained by qRT-PCR. (B) Protein expression of RANKL was detected by western blot, and b-tubulin served as a control. (C) TRAP-staining of BMMs cultured with conditioned media from MLO-Y4 cells; osteoclasts were identified as TRAP positive cells containing three or more nuclei (TRAP þ MNCs). “N/S-CM”, “N/SD-CM”, “H/S-CM”, “H/SD þ exo-CM” and “H/SD-CM” represented BMMs cultured with conditioned media from N/S treated MLO-Y4 cells, N/SD treated MLO-Y4 cells, H/S treated MLO-Y4 cells, H/SD þ exo treated MLO-Y4 cells and H/SD treated MLO-Y4 cells, respectively. All results are presented as the mean ± S.D. of at least three independent experiments. Analysis of variance; *P < 0.05 vs. N/S group; #P < 0.05 vs. H/SD group.
obviously inhibited excessive production of ROS stimulated by H/ SD. Apart from ROS, activation of mitochondrial-dependent apoptotic pathways is also reported to be responsible for cell death under hypoxia and ischemia stimulation [9]. A remarkable decrease in anti-apoptotic protein Bcl-2 is present in MLO-Y4 cells under a hypoxic-ischemic environment, while pro-apoptotic protein Bax appears in a contrary trend [28]. Our results also displayed the same trend for Bcl-2, and Bax expression at mRNA levels in MLO-Y4 cells in H/SD conditions. More importantly, ADSCs-exo could significantly reduce H/SD-induced downregulation of Bcl-2 and repress H/SD-induced upregulation of Bax. Moreover, factors including ROS and the Bcl-2 family of proteins may serve as upstream stimuli, which activate the mitochondria-dependent
apoptotic factors. Our results demonstrated ADSCs-exo effectively alleviated the release of cytochrome c, and the activation of caspase 9 and caspase-3 under H/SD conditions, which was similar to previous findings that the combination of ADSCs and ADSCs-exo can protect cardiomyocytes against I/R injury concomitant with downregulation of mitochondrial cytochrome c, Bax, cleaved caspase 3 and cleaved PARP [29]. In addition to inducing apoptosis, the H/SD treatment affected the expression of RANKL/OPG as well. Data have shown that apoptotic osteocytes may exhibit enhancement of osteoclastogenesis by elevated RANKL expression [6,30]. Increased RANKL, as well as decreased OPG, is reported by Domazetovic in MLO-Y4 cells for 24 h serum deprivation. It has been confirmed that the RANKL/ OPG ratio plays a vital role in regulating bone formation and bone
Please cite this article as: L. Ren et al., Adipose mesenchymal stem cell-derived exosomes ameliorate hypoxia/serum deprivation-induced osteocyte apoptosis and osteocyte-mediated osteoclastogenesis in vitro, Biochemical and Biophysical Research Communications, https:// doi.org/10.1016/j.bbrc.2018.11.109
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resorption [18]. In the current study, MLO-Y4 cells displayed a significant increase in RANKL levels and decreased OPG levels when cultured under H/SD conditions for 24 h. Particularly, the elevation of RANKL/OPG at the mRNA level and RANKL expression at the protein level were obviously reverted by ADSCs-exo under H/SD conditions, suggesting that exosomes may affect MLO-Y4-triggered osteoclastogenesis by regulating RANKL expression and the RANKL/ OPG ratio. As expected, less formation of multinucleated osteoclasts was found when BMMs were cultured with the condition medium from exosome treated osteocytes, indicating that ADSCsexo efficiently diminish osteocyte-mediated osteoclastogenesis under H/SD conditions. In conclusion, our study demonstrated, for the first time, that ADSCs-exo could effectively inhibit H/SD induced osteocyte apoptosis and osteocyte-mediated osteoclastogenesis. The antiapoptotic effects of ADSCs-exo were attained through the upregulation of Bcl-2/Bax, the suppression of ROS, cytochrome c generation and caspase-3, caspase-9 activation. Meanwhile, the inhibition of osteocyte-mediated osteoclastogenesis was obtained mainly via downregulation of the expression of RANKL. Animals studies should be required to further confirm the therapeutic effect of ADSCs for age-related bone diseases. Conflicts of interest The authors declare that they have no conflicts of interest. Acknowledgements This work was supported by the National Natural Science Foundation of China (Grant No. 81570955). This work was supported by Guangzhou Science and Technology and Innovation Commission (Grant No. 201510010222). Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2018.11.109. References [1] K.M. Nicks, S. Amin, E.J. Atkinson, B.L. Riggs, L.J. Melton, S. Khosla, Relationship of age to bone microstructure independent of areal bone mineral density, J. Bone Miner Res. 27 (2012) 637e644. https://doi.org/10.1002/jbmr.1468. [2] R.L. Jilka, C.A. O'Brien, The role of osteocytes in age-related bone loss, J. Curr. Osteoporos. Rep. 14 (2016) 16e25. https://doi.org/10.1007/s11914-016-02970. [3] D.M. Joiner, R.J. Tayim, J.D. McElderry, M.D. Morris, S.A. Goldstein, Aged male rats regenerate cortical bone with reduced osteocyte density and reduced secretion of nitric oxide after mechanical stimulation, J. Calcif. Tissue Int. 94 (2014) 484e494. https://doi.org/10.1007/s00223-013-9832-5. [4] T. Nakashima, M. Hayashi, T. Fukunaga, K. Kurata, M. Oh-Hora, J.Q. Feng, L.F. Bonewald, T. Kodama, A. Wutz, E.F. Wagner, J.M. Penninger, H. Takayanagi, Evidence for osteocyte regulation of bone homeostasis through RANKL expression, J. Nat. Med. 17 (2011) 1231e1234. https://doi.org/10.1038/nm. 2452. [5] S.A. Al-Dujaili, E. Lau, H. Al-Dujaili, K. Tsang, A. Guenther, L. You, Apoptotic osteocytes regulate osteoclast precursor recruitment and differentiation in vitro, J. Cell Biochem. 112 (2011) 2412e2423. https://doi.org/10.1002/jcb. 23164. [6] H.M. Davis, R. Pacheco-Costa, E.G. Atkinson, L.R. Brun, A.R. Gortazar, J. Harris, M. Hiasa, S.A. Bolarinwa, T. Yoneda, M. Ivan, A. Bruzzaniti, T. Bellido, L.I. Plotkin, Disruption of the Cx43/miR21 pathway leads to osteocyte apoptosis and increased osteoclastogenesis with aging, J. Aging Cell. 16 (2017) 551e563. https://doi.org/10.1111/acel.12586. [7] K. Kobayashi, H. Nojiri, Y. Saita, D. Morikawa, Y. Ozawa, K. Watanabe, et al., Mitochondrial superoxide in osteocytes perturbs canalicular networks in the setting of age-related osteoporosis, J. Sci. Rep. 5 (2015) 9148. https://doi.org/ 10.1038/srep09148. [8] A.E. Tami, P. Nasser, O. Verborgt, M.B. Schaffler, M.L.K. Tate, The role of interstitial fluid flow in the remodeling response to fatigue loading, J. Bone Miner Res. 17 (2002) 2030e2037. https://doi.org/10.1359/jbmr.2002.17.11.
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Please cite this article as: L. Ren et al., Adipose mesenchymal stem cell-derived exosomes ameliorate hypoxia/serum deprivation-induced osteocyte apoptosis and osteocyte-mediated osteoclastogenesis in vitro, Biochemical and Biophysical Research Communications, https:// doi.org/10.1016/j.bbrc.2018.11.109