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SNHG9, delivered by adipocyte-derived exosomes, alleviates inflammation and apoptosis of endothelial cells through suppressing TRADD expression
Journal Pre-proof SNHG9, delivered by adipocyte-derived exosomes, alleviates inflammation and apoptosis of endothelial cells through suppressing TRADD expression Yanbin Song, Hua Li, Xiaoyue Ren, Hongmei Li, Chuanjie Feng PII:
S0014-2999(20)30069-8
DOI:
https://doi.org/10.1016/j.ejphar.2020.172977
Reference:
EJP 172977
To appear in:
European Journal of Pharmacology
Received Date: 17 December 2019 Revised Date:
18 January 2020
Accepted Date: 29 January 2020
Please cite this article as: Song, Y., Li, H., Ren, X., Li, H., Feng, C., SNHG9, delivered by adipocytederived exosomes, alleviates inflammation and apoptosis of endothelial cells through suppressing TRADD expression, European Journal of Pharmacology (2020), doi: https://doi.org/10.1016/ j.ejphar.2020.172977. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier B.V.
SNHG9, delivered by adipocyte-derived exosomes, alleviates inflammation and apoptosis of endothelial cells through suppressing TRADD expression Yanbin Song 1, &, Hua Li 2, &, Xiaoyue Ren 3, Hongmei Li 2, Chuanjie Feng 4 1
Department of Cardiology, Yan’an University Affiliated Hospital, Yan’an, 716000,
China. 2
Department of Obstetrics, Yan’an University Affiliated Hospital, Yan’an, 716000,
China. 3
Department of Oncology, Yan’an University Affiliated Hospital, Yan’an, 716000,
China. 4
Emergency Department, Yan’an University Affiliated Hospital, Yan’an, 716000,
China. &
These authors contributed equally to this work.
* Corresponding author: Chuanjie Feng Emergency Department, Yan’an University Affiliated Hospital, No. 43 of North Street, Yan’an, 716000, China. Tel: +86-0911-2881097. Email: [email protected]
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Abstract Exosomes are membrane-derived vesicles and play a critical role in cell signaling by transferring RNAs and proteins to target cells through fusion with the cell membrane. Long non-coding RNA-small nucleolar RNA host gene 9 (lncRNA-SNHG9) was proven to be an important element in lncRNA–mRNA interaction networks during adipocyte differentiation, suggesting its potential involvement in the development of obesity, an important risk factor of cardiovascular and cerebrovascular endothelial dysfunction. However, the role of lncRNA-SNHG9 within the exosome in endothelial dysfunction of obese patients is largely unknown. In this study, we proved that adipocytes-derived exosomal SNHG9 were downregulated in obese persons and further decreased in obese individuals with endothelial dysfunction. Functional experimentations demonstrated that adipocytes-derived exosomal SNHG9 alleviated inflammation and apoptosis in endothelial cells. Bioinformatic analysis revealed that there was a potential interaction between SNHG9 and the TNF receptor type 1-associated death domain protein (TRADD) mRNA. Then, RNA-binding protein immunoprecipitation assay based on Ago2 antibody and ribonuclease protection assay demonstrated that exosomal SNHG9 directly bound to a specific region in TRADD mRNA sequence and formed an RNA dimeric inducible silencing complex. Moreover, knockdown of TRADD markedly inhibited inflammation and apoptosis in human umbilical vein endothelial cells (HUVECs), whereas overexpression of TRADD dramatically neutralized the
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protective effect of exosomal SNHG9 on epithelial dysfunction. Therefore, SNHG9 could prevent endothelial dysfunction in obese patients by suppressing inflammation and apoptosis, indicating that SNHG9 may be a potential therapeutic target for obese patients with endothelial dysfunction. Key Words: Adipocytes-derived exosomes; SNHG9; endothelial dysfunction; TRADD mRNA; Ago2-dependent RNA-RNA interaction
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1. Introduction Obesity is an important risk factor of vascular endothelial dysfunction (Deng et al., 2011; Lee et al., 2010), for its trigger on local or systemic inflammation (Jensen et al., 2013; Lumeng and Saltiel, 2011). The rate of global overweight and obesity in adolescence increase rapidly and has attracted a widespread concern. About 75%-80% of obese adolescents are still obese after adulthood for lacking of effective treatment measures, a high proportion of whom develop into persistent cardiovascular and cerebrovascular diseases (Guo et al., 2019; Peeters et al., 2003). Therefore, it is urgent to clarify the underlying mechanism of the endothelial dysfunction during obesity and explore some new intervention targets. Exosomes are nano-sized vesicles released into the extracellular environment by different cell types, including adipose-derived stem cells(Clotilde et al., 2009). Exosomes contain not only protein fragments, but also mRNAs, microRNAs and lncRNAs, and they are involved in a large amount of physiological processes and disease pathogenesis (Podbielska et al., 2016; Rong et al., 2014; Visovatti et al., 2012). Recent studies revealed that adipocytes-derived exosomes promoted vascular endothelial growth factor C-dependent lymph angiogenesis and have a critical role in lymphatic endothelial cells (Bradley, 2008; Sáez et al., 2018). In addition, adipocytes-derived exosomes had been shown to play a critical role in inflammation and fibrosis (Ferrante et al., 2015; Lazar et al., 2016). In obesity, adipocytes-derived exosomes may carry proinflammatory factors and transport them into remote cell
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types, thus interacting with receptor cells and exerting the proinflammatory effect (Giovanni et al., 2010; Lässer et al., 2011). Increasing evidence verified noncoding RNAs (including lncRNAs) enriched in exosomes might affect the function of exosomes by regulating its production and degradation, and they were widely used as predictors in the diseases (Boukouris and Mathivanan, 2015; Keller et al., 2011; Xue et al., 2017). Small nucleolar RNA host gene (SNHG) family is a group of lncRNAs and contributes to the regulation process of endothelial function and lipid metabolism. Previous studies revealed that lncRNA-SNHG16 affected synthesis of desaturase involved in lipid metabolism, and enzyme synthesis was regulated by the Wnt pathway (Christensen et al., 2016; Dong et al., 2018). A latest study also reported that overexpression of SNHG5 inhibited apoptosis and caused an increase of cell population at the S phase in HTR-8/SVneo cells(Liu et al., 2018). LncRNA-SNHG9 has been proven to be an important element in lncRNA–mRNA interaction networks during adipocyte differentiation, suggesting its potential involvement in the development of obesity, an important risk factor of diseases related to cardiovascular and cerebrovascular endothelial dysfunction (Chen et al., 2019), such as strokes, coronary artery diseases, peripheral arteria diseases and high blood pressure, for its trigger on local and/or systemic inflammation and stress responses. In this study, the exact role of lncRNA-SNHG9 within exosomes in endothelial dysfunction in obese patients is investigated. We explored the expression levels of exosomal SNHG9 in normal weight individuals, overweight but healthy
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individuals, and obesity individuals with endothelial dysfunction. The effect of adipocytes-derived exosomal SNHG9 on endothelial function also was investigated in human umbilical vein endothelial cells (HUVECs). To further illuminate the mechanism of exsomal SNHG9 in regulating endothelial function, HUVECs were treated with corresponding exosomes. Using RNA-binding protein immunoprecipitation assay and ribonuclease protection assay, we further confirmed the underlying mechanism responsible for exosomal SNHG9 in response to endothelial dysfunction. 2. Materials and Methods 2.1. Blood samples collection Blood samples were collected from adolescents, aged 7-14 years, from Aug 2017 to Dec 2018 at Yan’an University Affiliated Hospital (Yan’an, China). Among them, three independent cohorts were grouped. The control group was collected from healthy adolescents, body mass index (BMI) 18.5-23.99. The obese group was collected from healthy adolescents, BMI≥24. Another group was obese patients (BMI ≥ 24) accompanied by endothelial dysfunction, including diabetes (DB), coronary artery disease (CAD), peripheral arteria disease (PAD) or high blood pressure (HBP). The study was approved by the Ethics Committee of Yan’an University Affiliated Hospital (Approval number: YAUAH-2017056), and informed assents and consents were obtained from all subjects and legal guardians respectively. 2.2. Isolation of exosome-like vesicles
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Exosomes were separately isolated from plasmas or cell supernatants using Exoquick-TC (System Biosciences, Mountain View, CA) according to the manufacturer’s instruction. Extracted exosomes were characterized by Zetasizer Nano ZSP (Malvern Instruments, Malvern, U.K.) and Western blotting and stored in PBS at -80℃. 2.3. Cell culture and treatment Adipose-derived stem cells (ADSCs) and Human umbilical vein endothelial cells (HUVECs) were purchased from Cell Bank of Chinese Academy of Sciences (Shanghai, China). Cells were cultivated in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum (FBS) at 37°C, 5% CO2. ADSCs at the third passage were used for the experiments. Cells were plated in 24-well plates and transfected with Lipofectamine®2000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Overexpression of SNHG9 was achieved using Adenoviru-SNHG9 (Ad-SNHG9) transfection, with an empty vector as a control. Total protein samples were collected at different time points after transfection. 2.4. RNA extraction and RT-qPCR Total RNAs were extracted from cells by using TRIzol® reagent (Takara Biotechnology, Dalian, China), and complementary DNA (cDNA) was generated by using a Reverse Transcription Kit (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocol. Briefly, RNAs from each sample were reversely transcribed
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into cDNA with gene-specific RT primers. Then cDNAs were incubated at 95℃ for 10 min, followed by 35 cycles of 95℃ for 15 s, 58℃ for 30 s, and 72℃ for 30 s. Relative gene expression levels were calculated using the 2−∆∆Ct method and normalized to GAPDH. Each sample was analyzed in triplicate. 2.5. Western blotting The cells were seeded on 60-mm cell culture dishes and transfected with related plasmid vectors. After transfection for 48 h, total proteins were collected from cells with lysis buffer that had been added with PMSF (Bio-Rad, Hercules, CA, USA). Afterwards, concentration of proteins was measured by the BCA method. Then lysates were denatured with loading buffer at 100°C for 5 min. When dropping to room temperature, 25 µg protein was loaded on SDS-PAGE (12%) and transferred to PVDF membrane (Millipore, Boston, MA, USA), blocking with 10% skimmed milk powder. Then hybridizations with primary Abs were carried out for 1 h at room temperature. The protein-Ab complexes were detected by using peroxidase-conjugated secondary Abs (Boehringer Mannheim) and ECL (Luminata Forte, Millipore, USA). 2.6. Induction of adipogenic differentiation ADSCs at passage 3 were plated in 6-well plates at a confluence of 75%, cultured for 24 h and washed with PBS. Then cells were incubated with 2 ml of one of different culture media for 14 days. The media were used as follows: (1) α-minimal essential medium (α-MEM) supplemented with 10% FBS, as a negative control. (2)
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Adipogenic medium (α-MEM supplemented with 10% FBS, 1 mM DEX, 10 mM insulin, 200 mM indomethacin and 0.5 mM IBMX). The medium was changed every 3 days. After culture for 14 days, adipogenic differentiation was analyzed by Oil Red O (Sigma-Aldrich) staining. Then, Oil Red O in cells was extracted with 100% isopropanol for 15 min. The absorbance was measured at 510 nm under a spectrophotometer (Olympus, Tokyo, Japan). 2.7. RNA pull-down assay Biotinylated SNHG9 (SNHG9-probe) or biotinylated control (control-probe) was transfected into ADSCs. After 48h of transfection, the cells were lysed and incubated with M-280 streptavidin magnetic beads (Invitrogen, Carlsbad, CA, USA) at 25℃ for 2 h. Obtained compounds were centrifuged at 3000 g for 1 min. The bounded RNAs were then purified with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and analyzed by RT-PCR. 2.8. RNA-binding protein immunoprecipitation (RIP) assay RIP experiments were performed using the EZ-Magna RIP Kit (Millipore Corporation, Billerica, MA). Briefly, ADSCs were harvested and lysed. Then 100 ml of whole cell lysates were incubated with RIP buffer containing magnetic beads conjugated with human anti-Ago2 antibody (Millipore Corporation, Billerica, MA) or corresponding negative control IgG (Millipore Corporation, Billerica, MA). Then immunoprecipitated RNA associated with Ago2 antibody was isolated using TRIzol
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reagent. Subsequently the SNHG9 and TNF receptor type 1-associated death domain protein (TRADD) levels in the precipitates were investigated by RT-qPCR analysis. 2.9. Ribonuclease protection assay (RPA) The sample RNAs were mixed with the probe in the centrifuge tube containing 30 µl hybridization buffer. Incubate 5 min at 85℃ to denature RNA. Rapidly transfer to desired hybridization temperature and incubate for 12 h. Then add 350 µl ribonuclease digestion buffer to each hybridization reaction. Incubate 45 min at 30℃ to digest unprotected single-stranded RNA. Add 10 µl of 20% SDS and 2.5 µl of 20 mg/ml proteinase K. Incubate at 37℃ for 30 min to inactive RNase. Extract once with 400 µl phenol/chloroform/isoamyl alcohol, removing the aqueous phase to a microcentrifuge tube containing 1 µl of 10 mg/ml tRNA. Add 1 ml ethanol and precipitate. Dry pellet and redissolve in 3 to 5 µl RNA loading buffer. Incubate 5 min at 90°C to denature. Analyze on a denaturing polyacrylamide gel. 2.10. Statistical analysis Expression of exosomal SNHG9 in blood samples and transfection efficiency of Ad-SNHG9 in ADSCs were evaluated with t-test. The effects of exosomes on cell function were analyzed with one-way ANOVA. The data were expressed as means (from at least 3 repetitions) ± standard error of mean (S.E.M.). P < 0.05 was considered significant. 3. Results
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3.1. Expression of exosomal SNHG9 was downregulated in patients with endothelial dysfunction Blood samples containing exosomes were collected from subjects with a normal-weight, obese-only or obese accompanied by endothelial dysfunction. The detection results showed that exosomal SNHG9 expression from obese-only subjects were significantly lower than those from normal-weight subjects, and obese patients with endothelial dysfunction exhibited a further lower expression of SNHG9 in plasma exosomes (Fig. 1A). To examine the effect of exosomal SNHG9 on adipogenic differentiation, adenovirus vector Ad-SNHG9 was transfected into ADSCs. As shown in Fig. 1B, expression of SNHG9 was upregulated by Ad-SNHG9 in a dose-dependent manner. Subsequently, concentration of 1 µg was applied in the later studies. Then the effect of exosomal SNHG9 on adipogenic differentiation was investigated. Peroxisome proliferator-activated receptorγ2 (PPARγ2) is generally regarded as a key marker of adipogenesis, and its protein expression was detected by western blotting in our study. The results showed that no obvious change of PPARγ2 was revealed in the Ad-SNHG9 group compared with the control group (Fig. 1C). Oil Red O staining also showed that overexpression of SNHG9 makes no difference to generation of lipid droplets (Fig. 1D). 3.2. SNHG9-induced exosomes inhibited the endothelial dysfunction ADSCs were treated with adipogenic medium, and exosomes were separately collected at pre-treatment and post-treatment. Exosomes derived from
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SNHG9-induced ADSCs in days 14 were named as SNHG9-Day14-Exo. Exosomes derived from ADSCs transfection with control vector in day 0 were named as vector-Day0-Exo. Previous studies had shown that inflammation and apoptosis contributed to the endothelial dysfunction, so we analyzed the apoptosis rate and detected the expression of inflammatory markers including IL-1β, MMP-2, MMP-13. Meanwhile the NO enrichment and ET-1 expression, two indicators of endothelial dysfunction, were also investigated. The results showed that vector-Day0-Exosome group displayed higher SNHG9 expression compared with vector-Day0-cell group, and the expression levels of SNHG9 were much higher in SNHG9-Day14-cell group (Fig. 2A). Then the effects of exosomal SNHG9 on inflammation and apoptosis were studied. Our results showed that mRNA expression of IL-1β, MMP-2 and MMP-13 were inhibited by exosomes isolated from adipocytes transfecting with control vector, and a lower level of the inflammatory markers was observed in SNHG9-Day14-Exo group (Fig. 2B-2D). Subsequently we detected two biomarkers of endothelial dysfunction, NO production and ET-1 expression. ET-1 level exhibited a significant decrease in the vector-Day0-Exo group compared with control, and a further lower level of ET-1 was observed in SNHG9-Day14-Exo group (Fig. 2E). Correspondingly, treatment with vector-Day0-Exo resulted in a significantly increase on NO enrichment and eNOS expression. The fold change of NO content and eNOS protein from exosome-day14 group were further higher than exosome-Day0 group (Fig. 2F and 2G). As a key molecular mediator of endothelial dysfunction, p65 nuclear factor-κB
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(NF-κB) revealed a negative correlation with eNOS level (Fig. 2G). Furthermore, overexpression of SNHG9 alleviates inflammation-induced apoptosis (Fig. 2H). 3.3. SNHG9 binds to TRADD mRNA in an Ago2-dependent manner Bioinformatic analysis indicated that TRADD maybe interact with SNHG9, so we applied a biotin-avidin pull down system to test whether SNHG9 could pull down TRADD. Adipocytes were transfected with biotinylated TRADD, then harvested for RNA pull-down assay. Detecting results showed that SNHG9 could pull down TRADD using biotin-labelled-specific SNHG9 probe, which suggested that TRADD could directly bind to SNHG9 (Fig. 3A). Ago2, a key component of the miRNA-containing RISC complex, participated in amounts of molecular interactions. To investigate whether TRADD interacted with SNHG9 through Ago2, RIP assay was performed in adipocytes. Enrichment of transcripts in immunoprecipitated RNA was evaluated by RT-qPCR. As shown in Fig. 3B, TRADD and SNHG9 were both significantly enriched in the Ago2-immunoprecipitated RNA samples relative to input control in extracts of adipocyte. Further, we investigated the region which was targeted with SNHG9 in TRADD mRNA 3’UTR (Fig. 3C). RPA assay revealed that the product p1-p2 prevented SNHG9 primer from degradation, while the product p3-p4 could not make an effect in protecting SNHG9 (Fig. 3D). 3.4. Overexpression of TRADD abolished the protective effect of SNHG9 on endothelial dysfunction
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It had been proved that TRADD can interact with SNHG9 in HUVECs, we speculated that exosomal SNHG9 affects endothelial function by regulating expression of TRADD. Previous studies demonstrated that ox-LDL could effectively induce lipid accumulation and inflammatory reaction, so we applied ox-LDL treatment to HUVECs as an inflammatory model. The results showed that ox-LDL treatment resulted in an obvious increase in mRNA expression of TRADD, and the enhancement of TRADD expression was effectively neutralized by both TRADD knockdown and SNHG9-Day14-Exo exposure. Furthermore, overexpression of TRADD also attenuated the effect of SNHG9-Day14-Exo on TRADD expression in both mRNA and protein level, suggesting a negative correlation between TRADD and SNHG9 (Fig. 4A and 4B). SNHG9 induction or TRADD suppression both decreased the apoptosis rates; and the inhibiting effect of SNHG9-Day14-Exo on apoptosis was offset by the TRADD overexpression (Fig. 4C). Then we detected NF-κB signaling in our experiment models. As shown in Fig. 4D, NF-κB phosphorylation was inhibited by SNHG9 expression or TRADD suppression, and overexpression of TRADD effectively abolished the effect of SNHG9-Day14-Exo on activation of NF-κB pathway. IL-1β, MMP-2, MMP-13 and ET-1 were also investigated by Western blotting. The results showed that SNHG9 induction or TRADD suppression abated these pro-inflammatory genes expression (Fig. 4E-4H). In addition, NO enrichment regarded as a hallmark of endothelial dysfunction had been shown to decrease in cells
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treating with SNHG9-Day-Exo or TRADD siRNA, and overexpression of TRADD impaired the protective effect of SNHG9 on endothelial function (Fig. 4I). Generally, adipocytes release exosomes containing SNHG9. These SNHG9 molecules enter endothelial cells, bind with TRADD mRNA and form RNA-induced silencing complexes (Ago-2 protein as the core). Then, TRADD mRNA was degraded and its translation was blocked. Thus, SNHG9 alleviates inflammation and apoptosis of endothelial cells through binding with the TRADD mRNA and suppressing expression of the TRADD protein (Fig. 5). However, as we showed in Fig. 1, in obese patients, especially obese patients with vascular disorders, the adipocyte-derived exosomes contain much less SNHG9, which may be a reason for their endothelial dysfunction. 4. Discussion Adipocytes-derived exosomes function as messengers in varies of metabolic homeostasis, including reduced obesity attenuated adipose inflammation, and improved insulin sensitivity (Zhao et al., 2018). Previous studies found that some adipogenesis-related genes (such as PPARγ2) existed in exosomes derived from 3T3-L1 adipocytes, and these adipocytes-derived exosomes also contained abundant miRNAs and lncRNAs (for example, miR-103 and lnc-RAP-11), which were dramatically up-regulated during adipocyte differentiation (Li et al., 2015; Rumiko et al., 2010). LncRNA-SNHG9 had been revealed to be involved in the adipocyte differentiation, suggesting its potential involvement in the development of obesity
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(Chen et al., 2019). Herein, we found that adipocyte-derived exosomal SHNG9 were downregulated in obese persons with endothelial dysfunction. It is coincidence with the previous experiments, suggesting that adipocyte-derived exosomes engender beneficial effects on lipid metabolism. Moreover, our finding that SNHG9-Day14-cell extracts displayed a higher concentration of SNHG9 than SNHG9-Day14-Exosome may appear counterintuitive. On the contrary, this is actually an expected finding given that induction of adipogenic differentiation promoted the generation of lncRNA-SNHG9, and limited membrane transport was unable to deliver these molecules, leading to accumulation in cellular content. Inflammatory environments promote the release of exosomes that have mainly pro-inflammatory properties, and they are capable of affecting angiogenesis (Chistiakov et al., 2016; Roig-Arcos et al., 2017). Previous studies demonstrated that exosomes from blood circulation could facilitate angiogenesis and had an inhibition effect on HUVEC migration and tube formation (Chang et al., 2018; Jia et al., 2018). Moreover, inflammatory agents (such as soluble endoglin) contained in exosomes may participate in this process (Chang et al., 2018). In our current study, we demonstrated that exosomal-SNHG9 effectively inhibited production of pro-inflammatory factors, meanwhile enhanced production of NO, thus preventing HUVECs from endothelial dysfunction. Adipocytes-derived exosomal SNHG9 contributed to endothelial dysfunction by affecting inflammation, which was different from endothelial dysfunction induced by abundant soluble endoglin expression within
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exosomes(Chang et al., 2018). Our data showed that adipocytes-derived exosomes can deleteriously affect endothelial function by inhibiting the expression of IL-1β, MMP2 and MMP-13. TRADD functions as signal transmission in response to stimulus factors that has been identified to regulate inflammation and apoptosis by directly binding to the death domain of TNFR1 (Ihnatko and Kubes, 2007; Yelena L and Liu, 2012) . TRADD mediates the pathway downstream of TNFR1, including NF-κB, caspase and MAP kinase pathways, subsequently affects apoptosis and process of inflammation (Chang et al., 2017; Yelena L and Liu, 2012). Previous studies demonstrated that TRADD participated in the TNFα-induced apoptosis, and elevated expression of TRADD promoted the apoptosis in embryo cecum epithelial cells (Chang et al., 2017; Xu et al., 2017). In our present study, TRADD was found to be a target of exosomal SNHG9, and exosomal SNHG9 alleviated inflammation and apoptosis of endothelial cells through suppressing TRADD expression. In addition, p65 NF-κB, the marker protein of inflammation, was also negatively associated with the expression of TRADD. Our results were consistent with researches in embryo cecum epithelial cells and further confirmed the positive relativity between TRADD expression and endothelial function. These results suggest that pro-inflammatory and pro-apoptotic effects of TRADD maybe widely exist in various epithelial cells, and TRADD is a central signal linker in NF-κB signaling pathway.
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In conclusion, our results demonstrated that adipocytes-derived exosomal SNHG9 was lowly expressed in obese patients and further lower in individuals with epithelial dysfunction. ADSCs-derived exosomal SNHG9 inhibited the apoptosis and induced an anti-inflammatory effect on HUVECs. Overexpression of TRADD could effectively eliminate the protective effect of SNHG9 on epithelial dysfunction. Mechanism researches illustrated that TRADD is a target of SNHG9, NF-κB and AGO2 coordinately mediate SNHG9-induced biological functions in HUVECs. Taken together, our study revealed that SNHG9 directly bound to TRADD, activates the NF-κB signaling, thus inhibits the expression of inflammatory cytokines and apoptosis, eventually fulfills its protective effect on endothelial function. Conflict of Interests The authors declare that there is no conflict of interest regarding the publication of this paper. Acknowledgments This study was supported by the National Natural Science Foundation of China (No. 81760069 and 81960080), the Key Research and Development Plan of Shaanxi Province in China (No. 2018SF-116), the Health Care Project of Yan’an (No.2018KS-16) and the Project for Yan'an Science and Technology Innovation Team (No.H0206) in China.
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Lumeng, C.N., Saltiel, A.R., 2011. Inflammatory links between obesity and met abolic disease. J CLIN INVEST. 121, 2111-2117. https://doi.org/10.1172/J CI57132. Peeters, A., Barendregt, J., Willekens, F., Mackenbach, J., Al Mamun, A., Bon neux, L., 2003. Obesity in adulthood and its consequences for life expe ctancy: a life-table analysis. ANN INTERN MED. 138, 24-33. https://doi .org/10.7326/0003-4819-138-1-200301070-00008 Podbielska, M., Szulc, Z.M., Kurowska, E., Hogan, E.L., Bielawski, J., Bielaws ka, A., Bhat, N.R., 2016. Cytokine-induced release of ceramide-enriched exosomes as a mediator of cell death signaling in an oligodendrogliom a cell line. J LIPID RES. 57, 2028-2039. https://doi.org/10.1194/jlr.m070 664 Roig-Arcos, J., López-Malo, D., Díaz-Llopis, M., Romero, F.J., 2017. Exosomes derived from stimulated monocytes promote endothelial dysfunction and inflammation in vitro. ANN TRANSL MED. 5, 258-260. http://dx.doi.o rg/10.21037/atm.2017.03.101. Rong, J., Zhuang, Z.W., Zhang, J., Lanahan, A.A., Kyriakides, T., Sessa, W.C., Simons, M., 2014. Angiopoietin-2 Secretion by Endothelial Cell Exoso mes. J BIOL CHEM. 289, 510-519. https://doi.org/10.1074/jbc.m113.5068 99.
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Rumiko, O., Chie, T., Masahiro, S., Haruka, N., Kazuto, S., Katsuzumi, O., Yo shimi, N., Naohito, A., 2010. Adipocyte-derived microvesicles contain R NA that is transported into macrophages and might be secreted into blo od circulation. BIOCHEM BIOPH RES CO. 398, 723-729. https://doi.org /10.1016/j.bbrc.2010.07.008. Sáez, T., De, V.P., Sobrevia, L., Faas, M.M., 2018. Is there a role for exosome s in foetoplacental endothelial dysfunction in gestational diabetes mellitus ? PLACENTA. 61, 48-54. https://doi.org/10.1016/j.placenta.2017.11.007. Visovatti, S.H., Hyman, M.C., Diane, B., Richard, N., Mclaughlin, V.V., Pinsky, D.J., 2012. Increased CD39 nucleotidase activity on microparticles from patients with idiopathic pulmonary arterial hypertension. PLOS ONE. 7, e40829-e40839. https://doi.org/10.1371/journal.pone.0040829. Xu, Z.Y., Zheng, M.X., Zhang, L., Gong, X., Xi, R., Cui, X.Z., Bai, R., 2017. Dynamic expression of death receptor adapter proteins tradd and fadd i n Eimeria tenella-induced host cell apoptosis. POULTRY SCI. 96, 14381444. https://doi.org/10.3382/ps/pew496. Xue, M., Chen, W., Xiang, A., Wang, R., Chen, H., Pan, J., Pang, H., An, H., Wang, X., Hou, H., 2017. Hypoxic exosomes facilitate bladder tumor g rowth and development through transferring long non-coding RNA-UCA1 . MOL CANCER. 16, 143-. https://doi.org/10.1186/s12943-017-0714-8.
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Figure legends: Fig. 1. Exosomal SNHG9 was downregulated in blood samples of patients with endothelial dysfunction. (A) RT-qPCR was conducted to determine the expression of exosomal SNHG9 in blood samples collected from a normal-weight (n = 32), obese-only (n = 17) or obese accompanied by endothelial dysfunction (DB, n = 18; CAD, n = 23; PAD, n = 16 or HBP, n = 15) . (B) The infection efficiency of adenovirus vector Ad-SNHG9 into ADSCs was determined by RT-qPCR. (C) SNHG9 had no effect on protein expression of PPARγ2. (D) Formation of lipid droplet was analyzed by Oil Red O staining. The results were presented as the means ± S.E.M. according to 3 independent experiments in each sample or treatment cells. DB: diabetes, CAD: coronary artery disease, PAD: peripheral arteria disease, HBP: high blood pressure, SNHG9: Small nucleolar RNA host gene 9, PPARγ2: Peroxisome proliferator-activated receptorγ2, Ad-SNHG9: adenovirus SNHG9 overexpression
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vector. * Compared with vector, P < 0.05. # Compared with 0.1 µg group, P < 0.05. $
compared with 0.5 µg group, P < 0.05.
Fig. 2. Adipocytes-derived exosomal SNHG9 suppressed the endothelial dysfunction in HUVECs (n = 4). (A) Expression of SNHG9 was detected by RT-qPCR separately on pre- or post-adipogenic differentiation. *P < 0.05, **P < 0.01, ***P < 0.001. (B-D) SNHG9-induced exosomes decreased the expression of inflammatory cytokines. (E) ET-1 level was detected. (F) The production of NO was enhanced by treatment of exosomes derived ADSCs. (G) The expressions of p65 and eNOS were detected using western blot. (H) Apoptosis was inhibited by exosomes derived from ADSCs. *P < 0.05, **P < 0.01, ***P < 0.001. HUVECs: Human umbilical vein endothelial cells, ADSCs: Adipose-derived stem cells, vector-Day0-Exo: Exosomes derived from ADSCs transfection with control vector in day 0, SNHG9-Day14-Exo: Exosomes derived from SNHG9-induced ADSCs in days 14, Ox-LDL: Oxidized low density lipoprotein (ox-LDL) treatment to HUVECs as an inflammatory model. For B-G, *compared with control, P < 0.05. ## compared with vector-Day0-Exo group, P < 0.01. Fig. 3. SNHG9 binds to TRADD mRNA in an Ago2-dependent manner (n = 4). (A) The direct communication between SNHG9 and TRADD mRNA was identified by RNA pull-down. (B) The connection between TRADD and SNHG9 was further investigated by RIP assay. (C and D) The region of the combination was studied by RPA assay. The results were presented as the means ± S.E.M. TRADD: TNF receptor
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type 1-associated death domain protein, SNHG9: Small nucleolar RNA host gene 9. ***Compared with control probe or IgG, P < 0.001. Fig. 4. Overexpression of TRADD abolished the protective effect of exosomal SNHG9 on endothelial dysfunction (n = 4). (A) The mRNA expression of TRADD was analyzed by RT-qPCR after transfection with vectors. (B) The protein expression of TRADD was analyzed by Western blotting after transfection with vectors. (C) Apoptotic rates were investigated after treatment with exosomes. (D-G) The effect of TRADD on inflammatory cytokines was studied in HUVECs. (H and I) The productions of ET-1 and NO were detected in HUVECs. The results were presented as the means ± S.E.M. Ox-LDL: Oxidized low-density lipoprotein (ox-LDL) treatment to HUVECs as an inflammatory model, SNHG9-Day14-Exo: Exosomes derived from SNHG9-induced ADSCs in days 14, TRADD siRNA: TRADD small interfering RNA, Ad-TRADD: adenovirus TRADD overexpression vector. *Compared with control, P < 0.05. # Compared with ox-LDL, P < 0.05. Fig. 5.
A schematic diagram for the role of adipocyte-derived exosomal SNHG9 in
endothelial dysfunction (n = 4). Adipocyte-derived exosomal SNHG9 molecules enter endothelial cells, bind with TRADD mRNA and form RNA-induced silencing complexes (Ago-2 as the core). Then, TRADD mRNA was degraded and its translation was blocked. Thus, SNHG9 alleviates inflammation and apoptosis of endothelial cells. Blue arrows represent activating events. Red perpendicular bars represent inhibitory events.
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No conflict of interest exits in the submission of this manuscript, and manuscript is approved by all authors for publication. I would like to declare on behalf of my co-authors that the work described was original research that has not been published previously, and not under consideration for publication elsewhere, in whole or in part. All the authors listed in the submission have approved the manuscript. Yanbin Song, Hua Li, Xiaoyue Ren, Hongmei Li, Chuanjie Feng
S0014-2999(20)30069-8
DOI:
https://doi.org/10.1016/j.ejphar.2020.172977
Reference:
EJP 172977
To appear in:
European Journal of Pharmacology
Received Date: 17 December 2019 Revised Date:
18 January 2020
Accepted Date: 29 January 2020
Please cite this article as: Song, Y., Li, H., Ren, X., Li, H., Feng, C., SNHG9, delivered by adipocytederived exosomes, alleviates inflammation and apoptosis of endothelial cells through suppressing TRADD expression, European Journal of Pharmacology (2020), doi: https://doi.org/10.1016/ j.ejphar.2020.172977. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier B.V.
SNHG9, delivered by adipocyte-derived exosomes, alleviates inflammation and apoptosis of endothelial cells through suppressing TRADD expression Yanbin Song 1, &, Hua Li 2, &, Xiaoyue Ren 3, Hongmei Li 2, Chuanjie Feng 4 1
Department of Cardiology, Yan’an University Affiliated Hospital, Yan’an, 716000,
China. 2
Department of Obstetrics, Yan’an University Affiliated Hospital, Yan’an, 716000,
China. 3
Department of Oncology, Yan’an University Affiliated Hospital, Yan’an, 716000,
China. 4
Emergency Department, Yan’an University Affiliated Hospital, Yan’an, 716000,
China. &
These authors contributed equally to this work.
* Corresponding author: Chuanjie Feng Emergency Department, Yan’an University Affiliated Hospital, No. 43 of North Street, Yan’an, 716000, China. Tel: +86-0911-2881097. Email: [email protected]
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Abstract Exosomes are membrane-derived vesicles and play a critical role in cell signaling by transferring RNAs and proteins to target cells through fusion with the cell membrane. Long non-coding RNA-small nucleolar RNA host gene 9 (lncRNA-SNHG9) was proven to be an important element in lncRNA–mRNA interaction networks during adipocyte differentiation, suggesting its potential involvement in the development of obesity, an important risk factor of cardiovascular and cerebrovascular endothelial dysfunction. However, the role of lncRNA-SNHG9 within the exosome in endothelial dysfunction of obese patients is largely unknown. In this study, we proved that adipocytes-derived exosomal SNHG9 were downregulated in obese persons and further decreased in obese individuals with endothelial dysfunction. Functional experimentations demonstrated that adipocytes-derived exosomal SNHG9 alleviated inflammation and apoptosis in endothelial cells. Bioinformatic analysis revealed that there was a potential interaction between SNHG9 and the TNF receptor type 1-associated death domain protein (TRADD) mRNA. Then, RNA-binding protein immunoprecipitation assay based on Ago2 antibody and ribonuclease protection assay demonstrated that exosomal SNHG9 directly bound to a specific region in TRADD mRNA sequence and formed an RNA dimeric inducible silencing complex. Moreover, knockdown of TRADD markedly inhibited inflammation and apoptosis in human umbilical vein endothelial cells (HUVECs), whereas overexpression of TRADD dramatically neutralized the
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protective effect of exosomal SNHG9 on epithelial dysfunction. Therefore, SNHG9 could prevent endothelial dysfunction in obese patients by suppressing inflammation and apoptosis, indicating that SNHG9 may be a potential therapeutic target for obese patients with endothelial dysfunction. Key Words: Adipocytes-derived exosomes; SNHG9; endothelial dysfunction; TRADD mRNA; Ago2-dependent RNA-RNA interaction
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1. Introduction Obesity is an important risk factor of vascular endothelial dysfunction (Deng et al., 2011; Lee et al., 2010), for its trigger on local or systemic inflammation (Jensen et al., 2013; Lumeng and Saltiel, 2011). The rate of global overweight and obesity in adolescence increase rapidly and has attracted a widespread concern. About 75%-80% of obese adolescents are still obese after adulthood for lacking of effective treatment measures, a high proportion of whom develop into persistent cardiovascular and cerebrovascular diseases (Guo et al., 2019; Peeters et al., 2003). Therefore, it is urgent to clarify the underlying mechanism of the endothelial dysfunction during obesity and explore some new intervention targets. Exosomes are nano-sized vesicles released into the extracellular environment by different cell types, including adipose-derived stem cells(Clotilde et al., 2009). Exosomes contain not only protein fragments, but also mRNAs, microRNAs and lncRNAs, and they are involved in a large amount of physiological processes and disease pathogenesis (Podbielska et al., 2016; Rong et al., 2014; Visovatti et al., 2012). Recent studies revealed that adipocytes-derived exosomes promoted vascular endothelial growth factor C-dependent lymph angiogenesis and have a critical role in lymphatic endothelial cells (Bradley, 2008; Sáez et al., 2018). In addition, adipocytes-derived exosomes had been shown to play a critical role in inflammation and fibrosis (Ferrante et al., 2015; Lazar et al., 2016). In obesity, adipocytes-derived exosomes may carry proinflammatory factors and transport them into remote cell
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types, thus interacting with receptor cells and exerting the proinflammatory effect (Giovanni et al., 2010; Lässer et al., 2011). Increasing evidence verified noncoding RNAs (including lncRNAs) enriched in exosomes might affect the function of exosomes by regulating its production and degradation, and they were widely used as predictors in the diseases (Boukouris and Mathivanan, 2015; Keller et al., 2011; Xue et al., 2017). Small nucleolar RNA host gene (SNHG) family is a group of lncRNAs and contributes to the regulation process of endothelial function and lipid metabolism. Previous studies revealed that lncRNA-SNHG16 affected synthesis of desaturase involved in lipid metabolism, and enzyme synthesis was regulated by the Wnt pathway (Christensen et al., 2016; Dong et al., 2018). A latest study also reported that overexpression of SNHG5 inhibited apoptosis and caused an increase of cell population at the S phase in HTR-8/SVneo cells(Liu et al., 2018). LncRNA-SNHG9 has been proven to be an important element in lncRNA–mRNA interaction networks during adipocyte differentiation, suggesting its potential involvement in the development of obesity, an important risk factor of diseases related to cardiovascular and cerebrovascular endothelial dysfunction (Chen et al., 2019), such as strokes, coronary artery diseases, peripheral arteria diseases and high blood pressure, for its trigger on local and/or systemic inflammation and stress responses. In this study, the exact role of lncRNA-SNHG9 within exosomes in endothelial dysfunction in obese patients is investigated. We explored the expression levels of exosomal SNHG9 in normal weight individuals, overweight but healthy
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individuals, and obesity individuals with endothelial dysfunction. The effect of adipocytes-derived exosomal SNHG9 on endothelial function also was investigated in human umbilical vein endothelial cells (HUVECs). To further illuminate the mechanism of exsomal SNHG9 in regulating endothelial function, HUVECs were treated with corresponding exosomes. Using RNA-binding protein immunoprecipitation assay and ribonuclease protection assay, we further confirmed the underlying mechanism responsible for exosomal SNHG9 in response to endothelial dysfunction. 2. Materials and Methods 2.1. Blood samples collection Blood samples were collected from adolescents, aged 7-14 years, from Aug 2017 to Dec 2018 at Yan’an University Affiliated Hospital (Yan’an, China). Among them, three independent cohorts were grouped. The control group was collected from healthy adolescents, body mass index (BMI) 18.5-23.99. The obese group was collected from healthy adolescents, BMI≥24. Another group was obese patients (BMI ≥ 24) accompanied by endothelial dysfunction, including diabetes (DB), coronary artery disease (CAD), peripheral arteria disease (PAD) or high blood pressure (HBP). The study was approved by the Ethics Committee of Yan’an University Affiliated Hospital (Approval number: YAUAH-2017056), and informed assents and consents were obtained from all subjects and legal guardians respectively. 2.2. Isolation of exosome-like vesicles
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Exosomes were separately isolated from plasmas or cell supernatants using Exoquick-TC (System Biosciences, Mountain View, CA) according to the manufacturer’s instruction. Extracted exosomes were characterized by Zetasizer Nano ZSP (Malvern Instruments, Malvern, U.K.) and Western blotting and stored in PBS at -80℃. 2.3. Cell culture and treatment Adipose-derived stem cells (ADSCs) and Human umbilical vein endothelial cells (HUVECs) were purchased from Cell Bank of Chinese Academy of Sciences (Shanghai, China). Cells were cultivated in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum (FBS) at 37°C, 5% CO2. ADSCs at the third passage were used for the experiments. Cells were plated in 24-well plates and transfected with Lipofectamine®2000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Overexpression of SNHG9 was achieved using Adenoviru-SNHG9 (Ad-SNHG9) transfection, with an empty vector as a control. Total protein samples were collected at different time points after transfection. 2.4. RNA extraction and RT-qPCR Total RNAs were extracted from cells by using TRIzol® reagent (Takara Biotechnology, Dalian, China), and complementary DNA (cDNA) was generated by using a Reverse Transcription Kit (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocol. Briefly, RNAs from each sample were reversely transcribed
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into cDNA with gene-specific RT primers. Then cDNAs were incubated at 95℃ for 10 min, followed by 35 cycles of 95℃ for 15 s, 58℃ for 30 s, and 72℃ for 30 s. Relative gene expression levels were calculated using the 2−∆∆Ct method and normalized to GAPDH. Each sample was analyzed in triplicate. 2.5. Western blotting The cells were seeded on 60-mm cell culture dishes and transfected with related plasmid vectors. After transfection for 48 h, total proteins were collected from cells with lysis buffer that had been added with PMSF (Bio-Rad, Hercules, CA, USA). Afterwards, concentration of proteins was measured by the BCA method. Then lysates were denatured with loading buffer at 100°C for 5 min. When dropping to room temperature, 25 µg protein was loaded on SDS-PAGE (12%) and transferred to PVDF membrane (Millipore, Boston, MA, USA), blocking with 10% skimmed milk powder. Then hybridizations with primary Abs were carried out for 1 h at room temperature. The protein-Ab complexes were detected by using peroxidase-conjugated secondary Abs (Boehringer Mannheim) and ECL (Luminata Forte, Millipore, USA). 2.6. Induction of adipogenic differentiation ADSCs at passage 3 were plated in 6-well plates at a confluence of 75%, cultured for 24 h and washed with PBS. Then cells were incubated with 2 ml of one of different culture media for 14 days. The media were used as follows: (1) α-minimal essential medium (α-MEM) supplemented with 10% FBS, as a negative control. (2)
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Adipogenic medium (α-MEM supplemented with 10% FBS, 1 mM DEX, 10 mM insulin, 200 mM indomethacin and 0.5 mM IBMX). The medium was changed every 3 days. After culture for 14 days, adipogenic differentiation was analyzed by Oil Red O (Sigma-Aldrich) staining. Then, Oil Red O in cells was extracted with 100% isopropanol for 15 min. The absorbance was measured at 510 nm under a spectrophotometer (Olympus, Tokyo, Japan). 2.7. RNA pull-down assay Biotinylated SNHG9 (SNHG9-probe) or biotinylated control (control-probe) was transfected into ADSCs. After 48h of transfection, the cells were lysed and incubated with M-280 streptavidin magnetic beads (Invitrogen, Carlsbad, CA, USA) at 25℃ for 2 h. Obtained compounds were centrifuged at 3000 g for 1 min. The bounded RNAs were then purified with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and analyzed by RT-PCR. 2.8. RNA-binding protein immunoprecipitation (RIP) assay RIP experiments were performed using the EZ-Magna RIP Kit (Millipore Corporation, Billerica, MA). Briefly, ADSCs were harvested and lysed. Then 100 ml of whole cell lysates were incubated with RIP buffer containing magnetic beads conjugated with human anti-Ago2 antibody (Millipore Corporation, Billerica, MA) or corresponding negative control IgG (Millipore Corporation, Billerica, MA). Then immunoprecipitated RNA associated with Ago2 antibody was isolated using TRIzol
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reagent. Subsequently the SNHG9 and TNF receptor type 1-associated death domain protein (TRADD) levels in the precipitates were investigated by RT-qPCR analysis. 2.9. Ribonuclease protection assay (RPA) The sample RNAs were mixed with the probe in the centrifuge tube containing 30 µl hybridization buffer. Incubate 5 min at 85℃ to denature RNA. Rapidly transfer to desired hybridization temperature and incubate for 12 h. Then add 350 µl ribonuclease digestion buffer to each hybridization reaction. Incubate 45 min at 30℃ to digest unprotected single-stranded RNA. Add 10 µl of 20% SDS and 2.5 µl of 20 mg/ml proteinase K. Incubate at 37℃ for 30 min to inactive RNase. Extract once with 400 µl phenol/chloroform/isoamyl alcohol, removing the aqueous phase to a microcentrifuge tube containing 1 µl of 10 mg/ml tRNA. Add 1 ml ethanol and precipitate. Dry pellet and redissolve in 3 to 5 µl RNA loading buffer. Incubate 5 min at 90°C to denature. Analyze on a denaturing polyacrylamide gel. 2.10. Statistical analysis Expression of exosomal SNHG9 in blood samples and transfection efficiency of Ad-SNHG9 in ADSCs were evaluated with t-test. The effects of exosomes on cell function were analyzed with one-way ANOVA. The data were expressed as means (from at least 3 repetitions) ± standard error of mean (S.E.M.). P < 0.05 was considered significant. 3. Results
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3.1. Expression of exosomal SNHG9 was downregulated in patients with endothelial dysfunction Blood samples containing exosomes were collected from subjects with a normal-weight, obese-only or obese accompanied by endothelial dysfunction. The detection results showed that exosomal SNHG9 expression from obese-only subjects were significantly lower than those from normal-weight subjects, and obese patients with endothelial dysfunction exhibited a further lower expression of SNHG9 in plasma exosomes (Fig. 1A). To examine the effect of exosomal SNHG9 on adipogenic differentiation, adenovirus vector Ad-SNHG9 was transfected into ADSCs. As shown in Fig. 1B, expression of SNHG9 was upregulated by Ad-SNHG9 in a dose-dependent manner. Subsequently, concentration of 1 µg was applied in the later studies. Then the effect of exosomal SNHG9 on adipogenic differentiation was investigated. Peroxisome proliferator-activated receptorγ2 (PPARγ2) is generally regarded as a key marker of adipogenesis, and its protein expression was detected by western blotting in our study. The results showed that no obvious change of PPARγ2 was revealed in the Ad-SNHG9 group compared with the control group (Fig. 1C). Oil Red O staining also showed that overexpression of SNHG9 makes no difference to generation of lipid droplets (Fig. 1D). 3.2. SNHG9-induced exosomes inhibited the endothelial dysfunction ADSCs were treated with adipogenic medium, and exosomes were separately collected at pre-treatment and post-treatment. Exosomes derived from
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SNHG9-induced ADSCs in days 14 were named as SNHG9-Day14-Exo. Exosomes derived from ADSCs transfection with control vector in day 0 were named as vector-Day0-Exo. Previous studies had shown that inflammation and apoptosis contributed to the endothelial dysfunction, so we analyzed the apoptosis rate and detected the expression of inflammatory markers including IL-1β, MMP-2, MMP-13. Meanwhile the NO enrichment and ET-1 expression, two indicators of endothelial dysfunction, were also investigated. The results showed that vector-Day0-Exosome group displayed higher SNHG9 expression compared with vector-Day0-cell group, and the expression levels of SNHG9 were much higher in SNHG9-Day14-cell group (Fig. 2A). Then the effects of exosomal SNHG9 on inflammation and apoptosis were studied. Our results showed that mRNA expression of IL-1β, MMP-2 and MMP-13 were inhibited by exosomes isolated from adipocytes transfecting with control vector, and a lower level of the inflammatory markers was observed in SNHG9-Day14-Exo group (Fig. 2B-2D). Subsequently we detected two biomarkers of endothelial dysfunction, NO production and ET-1 expression. ET-1 level exhibited a significant decrease in the vector-Day0-Exo group compared with control, and a further lower level of ET-1 was observed in SNHG9-Day14-Exo group (Fig. 2E). Correspondingly, treatment with vector-Day0-Exo resulted in a significantly increase on NO enrichment and eNOS expression. The fold change of NO content and eNOS protein from exosome-day14 group were further higher than exosome-Day0 group (Fig. 2F and 2G). As a key molecular mediator of endothelial dysfunction, p65 nuclear factor-κB
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(NF-κB) revealed a negative correlation with eNOS level (Fig. 2G). Furthermore, overexpression of SNHG9 alleviates inflammation-induced apoptosis (Fig. 2H). 3.3. SNHG9 binds to TRADD mRNA in an Ago2-dependent manner Bioinformatic analysis indicated that TRADD maybe interact with SNHG9, so we applied a biotin-avidin pull down system to test whether SNHG9 could pull down TRADD. Adipocytes were transfected with biotinylated TRADD, then harvested for RNA pull-down assay. Detecting results showed that SNHG9 could pull down TRADD using biotin-labelled-specific SNHG9 probe, which suggested that TRADD could directly bind to SNHG9 (Fig. 3A). Ago2, a key component of the miRNA-containing RISC complex, participated in amounts of molecular interactions. To investigate whether TRADD interacted with SNHG9 through Ago2, RIP assay was performed in adipocytes. Enrichment of transcripts in immunoprecipitated RNA was evaluated by RT-qPCR. As shown in Fig. 3B, TRADD and SNHG9 were both significantly enriched in the Ago2-immunoprecipitated RNA samples relative to input control in extracts of adipocyte. Further, we investigated the region which was targeted with SNHG9 in TRADD mRNA 3’UTR (Fig. 3C). RPA assay revealed that the product p1-p2 prevented SNHG9 primer from degradation, while the product p3-p4 could not make an effect in protecting SNHG9 (Fig. 3D). 3.4. Overexpression of TRADD abolished the protective effect of SNHG9 on endothelial dysfunction
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It had been proved that TRADD can interact with SNHG9 in HUVECs, we speculated that exosomal SNHG9 affects endothelial function by regulating expression of TRADD. Previous studies demonstrated that ox-LDL could effectively induce lipid accumulation and inflammatory reaction, so we applied ox-LDL treatment to HUVECs as an inflammatory model. The results showed that ox-LDL treatment resulted in an obvious increase in mRNA expression of TRADD, and the enhancement of TRADD expression was effectively neutralized by both TRADD knockdown and SNHG9-Day14-Exo exposure. Furthermore, overexpression of TRADD also attenuated the effect of SNHG9-Day14-Exo on TRADD expression in both mRNA and protein level, suggesting a negative correlation between TRADD and SNHG9 (Fig. 4A and 4B). SNHG9 induction or TRADD suppression both decreased the apoptosis rates; and the inhibiting effect of SNHG9-Day14-Exo on apoptosis was offset by the TRADD overexpression (Fig. 4C). Then we detected NF-κB signaling in our experiment models. As shown in Fig. 4D, NF-κB phosphorylation was inhibited by SNHG9 expression or TRADD suppression, and overexpression of TRADD effectively abolished the effect of SNHG9-Day14-Exo on activation of NF-κB pathway. IL-1β, MMP-2, MMP-13 and ET-1 were also investigated by Western blotting. The results showed that SNHG9 induction or TRADD suppression abated these pro-inflammatory genes expression (Fig. 4E-4H). In addition, NO enrichment regarded as a hallmark of endothelial dysfunction had been shown to decrease in cells
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treating with SNHG9-Day-Exo or TRADD siRNA, and overexpression of TRADD impaired the protective effect of SNHG9 on endothelial function (Fig. 4I). Generally, adipocytes release exosomes containing SNHG9. These SNHG9 molecules enter endothelial cells, bind with TRADD mRNA and form RNA-induced silencing complexes (Ago-2 protein as the core). Then, TRADD mRNA was degraded and its translation was blocked. Thus, SNHG9 alleviates inflammation and apoptosis of endothelial cells through binding with the TRADD mRNA and suppressing expression of the TRADD protein (Fig. 5). However, as we showed in Fig. 1, in obese patients, especially obese patients with vascular disorders, the adipocyte-derived exosomes contain much less SNHG9, which may be a reason for their endothelial dysfunction. 4. Discussion Adipocytes-derived exosomes function as messengers in varies of metabolic homeostasis, including reduced obesity attenuated adipose inflammation, and improved insulin sensitivity (Zhao et al., 2018). Previous studies found that some adipogenesis-related genes (such as PPARγ2) existed in exosomes derived from 3T3-L1 adipocytes, and these adipocytes-derived exosomes also contained abundant miRNAs and lncRNAs (for example, miR-103 and lnc-RAP-11), which were dramatically up-regulated during adipocyte differentiation (Li et al., 2015; Rumiko et al., 2010). LncRNA-SNHG9 had been revealed to be involved in the adipocyte differentiation, suggesting its potential involvement in the development of obesity
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(Chen et al., 2019). Herein, we found that adipocyte-derived exosomal SHNG9 were downregulated in obese persons with endothelial dysfunction. It is coincidence with the previous experiments, suggesting that adipocyte-derived exosomes engender beneficial effects on lipid metabolism. Moreover, our finding that SNHG9-Day14-cell extracts displayed a higher concentration of SNHG9 than SNHG9-Day14-Exosome may appear counterintuitive. On the contrary, this is actually an expected finding given that induction of adipogenic differentiation promoted the generation of lncRNA-SNHG9, and limited membrane transport was unable to deliver these molecules, leading to accumulation in cellular content. Inflammatory environments promote the release of exosomes that have mainly pro-inflammatory properties, and they are capable of affecting angiogenesis (Chistiakov et al., 2016; Roig-Arcos et al., 2017). Previous studies demonstrated that exosomes from blood circulation could facilitate angiogenesis and had an inhibition effect on HUVEC migration and tube formation (Chang et al., 2018; Jia et al., 2018). Moreover, inflammatory agents (such as soluble endoglin) contained in exosomes may participate in this process (Chang et al., 2018). In our current study, we demonstrated that exosomal-SNHG9 effectively inhibited production of pro-inflammatory factors, meanwhile enhanced production of NO, thus preventing HUVECs from endothelial dysfunction. Adipocytes-derived exosomal SNHG9 contributed to endothelial dysfunction by affecting inflammation, which was different from endothelial dysfunction induced by abundant soluble endoglin expression within
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exosomes(Chang et al., 2018). Our data showed that adipocytes-derived exosomes can deleteriously affect endothelial function by inhibiting the expression of IL-1β, MMP2 and MMP-13. TRADD functions as signal transmission in response to stimulus factors that has been identified to regulate inflammation and apoptosis by directly binding to the death domain of TNFR1 (Ihnatko and Kubes, 2007; Yelena L and Liu, 2012) . TRADD mediates the pathway downstream of TNFR1, including NF-κB, caspase and MAP kinase pathways, subsequently affects apoptosis and process of inflammation (Chang et al., 2017; Yelena L and Liu, 2012). Previous studies demonstrated that TRADD participated in the TNFα-induced apoptosis, and elevated expression of TRADD promoted the apoptosis in embryo cecum epithelial cells (Chang et al., 2017; Xu et al., 2017). In our present study, TRADD was found to be a target of exosomal SNHG9, and exosomal SNHG9 alleviated inflammation and apoptosis of endothelial cells through suppressing TRADD expression. In addition, p65 NF-κB, the marker protein of inflammation, was also negatively associated with the expression of TRADD. Our results were consistent with researches in embryo cecum epithelial cells and further confirmed the positive relativity between TRADD expression and endothelial function. These results suggest that pro-inflammatory and pro-apoptotic effects of TRADD maybe widely exist in various epithelial cells, and TRADD is a central signal linker in NF-κB signaling pathway.
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In conclusion, our results demonstrated that adipocytes-derived exosomal SNHG9 was lowly expressed in obese patients and further lower in individuals with epithelial dysfunction. ADSCs-derived exosomal SNHG9 inhibited the apoptosis and induced an anti-inflammatory effect on HUVECs. Overexpression of TRADD could effectively eliminate the protective effect of SNHG9 on epithelial dysfunction. Mechanism researches illustrated that TRADD is a target of SNHG9, NF-κB and AGO2 coordinately mediate SNHG9-induced biological functions in HUVECs. Taken together, our study revealed that SNHG9 directly bound to TRADD, activates the NF-κB signaling, thus inhibits the expression of inflammatory cytokines and apoptosis, eventually fulfills its protective effect on endothelial function. Conflict of Interests The authors declare that there is no conflict of interest regarding the publication of this paper. Acknowledgments This study was supported by the National Natural Science Foundation of China (No. 81760069 and 81960080), the Key Research and Development Plan of Shaanxi Province in China (No. 2018SF-116), the Health Care Project of Yan’an (No.2018KS-16) and the Project for Yan'an Science and Technology Innovation Team (No.H0206) in China.
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Figure legends: Fig. 1. Exosomal SNHG9 was downregulated in blood samples of patients with endothelial dysfunction. (A) RT-qPCR was conducted to determine the expression of exosomal SNHG9 in blood samples collected from a normal-weight (n = 32), obese-only (n = 17) or obese accompanied by endothelial dysfunction (DB, n = 18; CAD, n = 23; PAD, n = 16 or HBP, n = 15) . (B) The infection efficiency of adenovirus vector Ad-SNHG9 into ADSCs was determined by RT-qPCR. (C) SNHG9 had no effect on protein expression of PPARγ2. (D) Formation of lipid droplet was analyzed by Oil Red O staining. The results were presented as the means ± S.E.M. according to 3 independent experiments in each sample or treatment cells. DB: diabetes, CAD: coronary artery disease, PAD: peripheral arteria disease, HBP: high blood pressure, SNHG9: Small nucleolar RNA host gene 9, PPARγ2: Peroxisome proliferator-activated receptorγ2, Ad-SNHG9: adenovirus SNHG9 overexpression
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vector. * Compared with vector, P < 0.05. # Compared with 0.1 µg group, P < 0.05. $
compared with 0.5 µg group, P < 0.05.
Fig. 2. Adipocytes-derived exosomal SNHG9 suppressed the endothelial dysfunction in HUVECs (n = 4). (A) Expression of SNHG9 was detected by RT-qPCR separately on pre- or post-adipogenic differentiation. *P < 0.05, **P < 0.01, ***P < 0.001. (B-D) SNHG9-induced exosomes decreased the expression of inflammatory cytokines. (E) ET-1 level was detected. (F) The production of NO was enhanced by treatment of exosomes derived ADSCs. (G) The expressions of p65 and eNOS were detected using western blot. (H) Apoptosis was inhibited by exosomes derived from ADSCs. *P < 0.05, **P < 0.01, ***P < 0.001. HUVECs: Human umbilical vein endothelial cells, ADSCs: Adipose-derived stem cells, vector-Day0-Exo: Exosomes derived from ADSCs transfection with control vector in day 0, SNHG9-Day14-Exo: Exosomes derived from SNHG9-induced ADSCs in days 14, Ox-LDL: Oxidized low density lipoprotein (ox-LDL) treatment to HUVECs as an inflammatory model. For B-G, *compared with control, P < 0.05. ## compared with vector-Day0-Exo group, P < 0.01. Fig. 3. SNHG9 binds to TRADD mRNA in an Ago2-dependent manner (n = 4). (A) The direct communication between SNHG9 and TRADD mRNA was identified by RNA pull-down. (B) The connection between TRADD and SNHG9 was further investigated by RIP assay. (C and D) The region of the combination was studied by RPA assay. The results were presented as the means ± S.E.M. TRADD: TNF receptor
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type 1-associated death domain protein, SNHG9: Small nucleolar RNA host gene 9. ***Compared with control probe or IgG, P < 0.001. Fig. 4. Overexpression of TRADD abolished the protective effect of exosomal SNHG9 on endothelial dysfunction (n = 4). (A) The mRNA expression of TRADD was analyzed by RT-qPCR after transfection with vectors. (B) The protein expression of TRADD was analyzed by Western blotting after transfection with vectors. (C) Apoptotic rates were investigated after treatment with exosomes. (D-G) The effect of TRADD on inflammatory cytokines was studied in HUVECs. (H and I) The productions of ET-1 and NO were detected in HUVECs. The results were presented as the means ± S.E.M. Ox-LDL: Oxidized low-density lipoprotein (ox-LDL) treatment to HUVECs as an inflammatory model, SNHG9-Day14-Exo: Exosomes derived from SNHG9-induced ADSCs in days 14, TRADD siRNA: TRADD small interfering RNA, Ad-TRADD: adenovirus TRADD overexpression vector. *Compared with control, P < 0.05. # Compared with ox-LDL, P < 0.05. Fig. 5.
A schematic diagram for the role of adipocyte-derived exosomal SNHG9 in
endothelial dysfunction (n = 4). Adipocyte-derived exosomal SNHG9 molecules enter endothelial cells, bind with TRADD mRNA and form RNA-induced silencing complexes (Ago-2 as the core). Then, TRADD mRNA was degraded and its translation was blocked. Thus, SNHG9 alleviates inflammation and apoptosis of endothelial cells. Blue arrows represent activating events. Red perpendicular bars represent inhibitory events.
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No conflict of interest exits in the submission of this manuscript, and manuscript is approved by all authors for publication. I would like to declare on behalf of my co-authors that the work described was original research that has not been published previously, and not under consideration for publication elsewhere, in whole or in part. All the authors listed in the submission have approved the manuscript. Yanbin Song, Hua Li, Xiaoyue Ren, Hongmei Li, Chuanjie Feng