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Monocyte-derived extracellular vesicles upon treated by palmitate promote endothelial migration and monocytes attachment to endothelial cells
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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Monocyte-derived extracellular vesicles upon treated by palmitate promote endothelial migration and monocytes attachment to endothelial cells Wanhao Gao 1, Xingchen Guo 1, Ying Wang, Dongdong Jian, Muwei Li* Department of Cardiology, People’s Hospital of Zhengzhou University, Zhengzhou University, Zhengzhou, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 December 2019 Accepted 28 December 2019 Available online xxx
Aim: High circulating free fatty acid (FFA) concentration has a critical role in the development of obesity associated vascular comorbidities. Ample previous findings revealed that FFA, especially saturated, induce endothelial dysfunction throught multiple mechanisms (summarized as lipotoxicity). As a mediator that transfers information among cells, extracellular vesicles(EVs) participate in pathologic processes of many diseases, including angiocardiopathy, insulin resistance, autoimmunity disease. However, how lipotoxicity changed the proportion of EVs secreted from monocytes, furthermore, the effect of the EVs exerts on endothelial cells, haven’t been demonstrated. Method: In our experience, differential ultracentrifugation was used to extract EVs from condition medium (CM) of THP-1 monocytes under given treatments. Then we co-incubated the EVs derived from palmitate-treated monocytes with HUVECs for 24 h, after which molecular and phenotypic assays were conducted. Result: Palmitate-treated monocytes EVs promote the production of adhesion associated proteins of endothelial cells, such as VCAM-1, ICAM-1. Meanwhile, palmitate-stimulation may play a promoter role in the pro-migration capacity of monocytes-EVs. In brief, EVs could be the new pathological junction between FFA and endothelial damage. © 2020 Elsevier Inc. All rights reserved.
Keywords: Extracellular vesicles Monocytes Obesity Saturated FFA Endothelial cells
1. Introduction Around the beginning of the twenty-first century, obesity has emerged as an enormous public health problem globally [1]. These patients who confront an increased risk to develop vascular diseases, especially, atherosclerosis [2]. One of the characteristic abnormalities in obesity is high circulating FFA concentration. Clinical studies and in vivo studies have shown that high FFA levels are associated with impairment of endothelium-dependent vasodilation [3], insulin resistance [4,5], myocardial infarction [6], stroke [7]. In addition, FFA affect gene expression of Vascular Smooth Muscle Cells [8], adipocytes [9], macrophages [10,11] and monocytes [12]. Although FFA was
* Corresponding author. E-mail addresses: [email protected], [email protected] (W. Gao), [email protected] (X. Guo), [email protected] (Y. Wang), 11418149@ zju.edu.cn (D. Jian), [email protected] (M. Li). 1 These authors contributed equally to this work.
considered as one of the pathological junctions between obesity and endothelial damage, mechanisms of the FFA-induced comorbidities are still incompletely understood. Extracellular vesicles(EVs), the membrane-bound structure of cells [13], can be broadly classified into 3 main classes: Exosomes(released upon the fusion of multi-vesicular bodies), Microvesicles(outward budding and fission of plasma membrane), Apoptotic bodies(released as blebs of apoptotic cells). Although first discovered in the late 1960s, EVs were stated as an excretion mechanism through which cells eliminate unnecessary cellular metabolic wastes. Until 2007, a finding [14] pointed out the ability of EVs exchange cargo(containing proteins, mRNAs, and microRNAs) from donor cells to acceptor cells and triggering phenotypic changes in the latter, interest of the scientific community was attracted substantially. However, current EVs purify protocols are difficult to obtain pure EVs subtype [15], which brings a challenge to EVs research. Therefore, according to physical properties, EVs were simply classified as sEV (small EV) and lEV (large EV) in various studies. MISEV [16] also suggests that the EVs without strict origin demonstration, shouldn’t be
https://doi.org/10.1016/j.bbrc.2019.12.095 0006-291X/© 2020 Elsevier Inc. All rights reserved.
Please cite this article as: W. Gao et al., Monocyte-derived extracellular vesicles upon treated by palmitate promote endothelial migration and monocytes attachment to endothelial cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.095
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named as exosomes or microvesicles, but distinguished by their size. EVs carry out their physiological functions in many pathophysiological situations, including immune responses [17],cardiovascular diseases [18] and cell-based therapy [19]. The recent finding also suggests [20], EVs play a significant role of mediator in endothelial cell function. Like most other cells, monocytes also release functional EVs [21]. Furthermore, monocyte-derived EVs diffuse in circulating blood, which greatly increased the possibility of uptaken by vascular endothelial cells. The relative proportion of released EVs is highly variable, which indicates the broader function EVs may possess. Therefore, we hypothesize that lipotoxicity induced monocytes EVs deliver molecules to endothelial cells, and participate in the development of endothelial dysfunction in obesity. 2. Material and methods 2.1. Antibodies and reagents Primary antibodies used for western blot included VCAM-1(Cat. No. ab134047), ICAM-1(Cat. No. ab109361), e-NOS(Cat. No. ab199956), p53(Cat. No. ab32389), Cyclin-D1(Cat. No. ab226977), TSG101(Cat. No. ab125011), Antibody HSP70(Cat. No. WL01019),Calnexin(Cat. No. WL03062) was purchased from Wanleibio(Shenyang, China), Antibody BAX (Cat. No. CY5059) was purchased from Abway(Beijing, China). E-selectin(Cat. No. 208941-AP), p65(Cat. No. 10745-1-AP) GAPDH(Cat. No. 10494-1-AP), beta-Actin(Cat. No. 20536-1-AP) were purchased from Proteintech Group (Proteintech, USA). Endothelial cell medium(ECM) was purchased from Sciencell(CA, USA); RPMI1640 and Fetal bovine serum (FBS) were purchased from Invitrogen(Carlsbad, USA). Saturated FFA palmitate (16: 0) and its solvent(Cat. No. SYSJ001) were purchased from Kunchuang biotechnology. RIPA buffer, SDS-PAGE Sample Loading Buffer, HRP-labeled Goat Anti-Rabbit/Mouse IgG, Coomassie Blue Staining Solution, Crystal Violet Staining Solution were purchased from Beyotime(Beijing, China). CFDA was purchased Solarbio(Beijing, China). Pierce BCA Protein Assay Kit was purchased from Thermofisher (Rockford, USA).
based on ultrafiltration to deplete EVs from FBS. Briefly, Ultra-15 Centrifugal Filter Devices (UFC910024, 100 kDa) were purchased from Merk Millipore(Darmstadt, Germany). EV-depleted FBS was prepared according to the studies of Kornilov et al. [22]. EVdepleted RPMI1640 medium was compound with 10% EVdepleted FBS. EVs isolation from monocytes CM: EVs were isolated and purified by classic differential ultracentrifugation [23], but with minor modifications. All the centrifugal steps described blew were conducted at 4 C. The CM of THP-1 monocytes was centrifuged at 600 g for 5 min to remove whole cells. And then centrifuged at 1200 g for 15 min to remove cell debris and apoptotic bodies. The resulting supernatant was centrifuged at 10,000 g for 20 min to remove lEVs. Finally, the supernatant was centrifuged in a Swinging-Bucket angle rotor (SW 32Ti, Beckman) at 100,000 g for 90 min to pellet sEVs. The pellet was resuspended in PBS and centrifuged again at 100,000 g for another 90 min. The final pellet was suspended in 100 ml PBS, and EVs were quantified by BCA assay (Thermo Scientific). EVs samples derived from THP-1 monocytes treated by palmitate (labeled as PA-EVs), Solvent (labeled as Solvent Control, SCEVs) or nothing (labeled as Normal Control, NC-EVs), were used in the subsequent analyses. EVs preparations were aliquoted and stored at 80 C and subjected to a single freeze-thaw. 2.4. Characterization of EVs Nanoparticle tracking analysis (NTA): The EVs concentration and size distribution was analyzed by NTA after diluting EVs preparations with PBS to obtain the optimal detection concentration. Three 40s videos were recorded and the data was analyzed using NTA software 3.0. Transmission electron microscopy (TEM): EVs samples isolated by ultracentrifugation were prepared for TEM and images described previously [24]. Samples were viewed with transmission EM using HT7800 TEM system(Hitachi Company) operating at 80 kV. Images were taken with 3295 2569 px image size. 2.5. EV treatments
2.2. Cell culture and stimulation THP-1 cells, obtained from American Type Culture Collection (Manassas, USA), were cultured in RPMI 1640 medium supplemented with 10% FBS, 100U/ml penicillin and 100 mg/ml streptomycin (all from Invitrogen, USA). Adjust the density of cell suspension ranged from 2 105 to 1 106 cells/mL during cell expansion. Prior to cell stimulation, collected THP-1 monocytes by centrifugal separation were suspended in medium containing EVs-depleted FBS (protocol described below), Then, to treat THP1 cell with saturated FFA palmitate (16: 0; 200 mM), Solvent control(equal volume with palmitate) or nothing for 24 h. Collecting conditioned media (CM) of different groups for isolation of EVs. Primary human umbilical vein endothelial cells (HUVECs), obtained from Sciencell (CA, USA), were maintained in endothelial cell medium containing 5%FBS, 2 mM glutamine, 100U/ml penicillin, and 100 mg/ml streptomycin. All the cells were cultured at 37 C and 5% CO2 in a humidified incubator. 2.3. EVs isolation Preparation of EV-depleted FBS: in order to minimize the effect of FBS EVs on downstream analysis. We use a protocol
HUVECs were plated on twelve-well plates for a maximum of 6 passages. Upon 60% confluence, HUVECs were treated with NCEVs(NC-Group), PA-EVs(PA-Group), SC-EVs(SC-Group) for 24 h, EVs was add in ECM of each treated group at a final concentration of 20 mg/ml. HUVECs treated with the same volume of PBS were used as an experimental control(Basal-Group), after this, HUVECs was used for protein extraction and other phenotypic assays. 2.6. Western blot analysis Western blotting was performed as described previously [25], Cells and EVs preparations were lysed with RIPA buffer at 4 C for 30 min, protease inhibitors were added in RIPA buffer in advance. After centrifuged at 12,000 g for 10 min at 4 C, lysed was collected and quantified by the BCA method. Use RIPA buffer to leveling the protein concentration. Protein preparations were denatured at 95 C for 10min in sample Loading Buffer. Equal solubilized proteins were added to polyacrylamide SDS gels. After transferred proteins onto polyvinylidene difluoride membranes (Millipore, USA), Membranes were incubated with Western Blocking Buffer for 60 min at room temperature and then soaked in indicated antibodies overnight at 4 C. After incubation, blots were washed three times in TBS-T for 10 min at RT. Blotting with a horseradish peroxidase-conjugated secondary antibody (diluted 1: 2000 in
Please cite this article as: W. Gao et al., Monocyte-derived extracellular vesicles upon treated by palmitate promote endothelial migration and monocytes attachment to endothelial cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.095
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TBST) for 1 h at room temperature and then visualizing by ECL Kit (Millipore, USA). quantification was performed using Image J software. 2.7. Monocyte attachment assay As described above,60%e80% confluent HUVECs were treated with PBS, NC-EVs, PA-EVs, SC-EVs in a 12-well plate, 20 mg/ml EVs was add in each treated group. 24 h later, the Attachment assay was conducted referring to an earlier report [26]. Briefly, removed the ECM in the wells and washed the residual medium by PBS twice times. THP-1 monocytes were stained by CFDA(Solarbio, China) for 30 min in advanced according to instruction. 1.0 ml suspension of the fluorescently-labeled THP-1 monocytes was added in each well containing a confluent monolayer of HUVECs. Co-incubating THP-1 monocytes and HUVECs for 1 h, after which, non-adherent THP-1 monocytes were removed by three times PBS washes. Five images from the area around the center of each well were captured by an inverted fluorescence microscope. The percentage of total image area covered by THP1 cells was measured by Image J software. 2.8. Scratch wound healing assay Scratch assays were performed as described previously [20]. Upon 90e100% confluence, the monolayer of HUVECs in 12-well plates was scratched with a sterile pipette (200ml). Then low-
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serum ECM (1% EVs-depleted FBS) was added to the wells and cells were stimulated with PBS,NC-EVs(20 mg/ml),PA-EVs(20 mg/ ml),SC-EVs (20 mg/ml) for 24 h, which same with above description. Cells were photographed on the marked position at 0, 6 12 24 h. Use image J to measure and correlated the remaining cell-free area to the initial scratched area (in percent). 2.9. Transwell migration assay Transwell assays were performed using 8.0-mm-pore transwell chambers (Corning, USA) with polycarbonate filters. The trial grouping method was the same as the Scratch wound healing assay but in EVs-depleted 5%FBS medium. Subsequently, collected HUVECs by centrifugal separation was resuspended and counted. Then 200 ml of HUVECs suspension (about 1 105/ml in ECM containing 1% FBS) was added in the upper chamber, whereas the lower chamber contained ECM supplemented with 10% FBS. After 12 or 24 h, the medium in the upper chamber was discarded. And then, using a cotton swab to abrase non-migrating cells from the upper surface of the membrane. Cells on the underside were fixed with 4% paraformaldehyde for 20 min and then stained with crystal violet staining solution for 10 min. Finally, washing and removed resident crystal violet three times by PBS. Counting the number of stained Migrating cells from four randomly chosen high-power fields ( 100) with a phasecontrast microscope (Olympus, Japan). All assays were performed in three separate sets of experiments.
Fig. 1. Characteristic of Extracellular Vesicle derived from THP-1 monocytes. (A)transmission electron micrograph revealing the typical morphology and size of EVs. (B) Western blot reveals the presence of the exosomal protein markers TSG101, HSP70. Proteins of THP-1 monocytes used as control group, identified GAPDH and calnexin undetectable in EVs preparations. (C) Coomassie Brilliant Blue staining of extracted proteins of THP-1 monocytes and EVs. NTA profiles confirmed the size distribution of the EVs preparations that secreted from normal THP-1 cell (D) or THP-1 cell treated with solvent-control (F) or palmitate(E). . (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Please cite this article as: W. Gao et al., Monocyte-derived extracellular vesicles upon treated by palmitate promote endothelial migration and monocytes attachment to endothelial cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.095
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2.10. Statistical analyses
to 75kda and no significant difference among three EVs groups.
Data are presented as mean ± SEM. *p < 0.05, **p < 0.01 and ***p < 0.001 by one-way ANOVA. A p-value < 0.05 was determined to be statistically significant. GraphPad software was used to perform all analyses and to construct all graphs.
3.2. The effect of monocytes EVs exert on endothelial cell inflammation
3. Result 3.1. Characterization of EVs EVs from THP-1 monocytes CM were isolated by ultracentrifugation and visualized by TEM after negative staining. As shown in Fig. 1A, isolated EVs possess typical round, oval or cup-shaped, lipid bilayer membranous vesicular morphology. Apart from the ultrastructure of EVs, TEM also reveals a size in a range of 30e150 nm in diameter sketchily. Furthermore, NTA profiles confirmed the size distribution of the EVs preparations, that the peak analysis(concentration)of EVs secreted from normal THP-1 cell (146.5 ± 2.28 nm, Fig. 1D) and THP-1 cell treated with palmitate (138.6 ± 2.12 nm, Fig. 1F) was similar to that treated with solventcontrol(142.2 ± 0.96 nm, Fig. 1E). Upon further characterization by Western blot, using TSG101 and HSP70 as Exosomal marker, Calnexin as an endoplasmic reticulum marker, GAPDH as a cytoplast marker. In Fig. 1B, the blots of TSG101 and HSP70 proteins reveals distinct traces, while small amounts of Calnexin and GAPDH proteins could be detected, which confirms that the EVs preparations were free of contaminating proteins. Coomassie Brilliant Blue staining(Fig. 1C) suggesting molecular weight distribution of EVs proteins separated on SDS-PAGE gels, whose peaks(concentration)range from 60kda
Monocytes attached to activated human vascular endothelium is the initial step of atherogenesis. To detect the effect of monocytes derived EVs exert on HUVECs, monocytes attachment assays were implemented initially. Co-incubating Fluorescent dyed and numerically equal THP-1 cells with HUVECs, which pretreated with NC-EVs, PA-EVs, SC-EVs or PBS for 24 h. As Fig. 2Eshown, PA-EVs treat groups exhibit a significantly increased monocytes attachment, compared to those observed for HUVECs didn’t treated with EVs. The trendy was inexistent among the other three groups. To confirm it, following treatment of HUVECs with monocytes EVs, Western blotting was used to detect the production of adhesion associated proteins involved in monocytes attachment. As Fig. 2A/B shown, the production of the protein: ICAM-1 and ICAM-1 was increased in HUVECs treated with PA-EVs compared to that of untreated HUVECs(Basal Group). No detectable change in NC-EVs nor SC-EVs Group. The level of E-selectin protein has no significant change in both PA-EVs and NC-EVs groups. Although slightly increasing trend was observed in SC-EVs groups, there is no statistical significance was obtained (P ¼ 0.1681). As a crucial regulation protein of inflammation signal, NF-ΚB also participates in the pro-inflammation function of EVs, as research reported earlier [27]. However, in our experiment, lipotoxicity induced monocytes derived EVs didn’t change the proteins level of p65 in HUVECS.
Fig. 2. The effect of monocytes EVs exert on endothelial cell inflammation Representative graph showing the ratio of (A) ICAM-1, (B) VCAM-1, (C) E-Selectin, (D) p65 expression in HUVECs following treatment with PBS(Basal), NC-EVs(NC), PA-EVs(PA), SC-EVs(SC). Representative blots of proteins are shown in Fig. 2G. (E)Monocytes-endothelial cells attachment assay and (F) Ratio of percentage area covered by monocytes to a confluent monolayer of HUVECs. Green dots are THP-1 monocytes stained with CFDA All data values represent the mean ± SEM of triple.independent experience *p < 0.05, **p < 0.01. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Please cite this article as: W. Gao et al., Monocyte-derived extracellular vesicles upon treated by palmitate promote endothelial migration and monocytes attachment to endothelial cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.095
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3.3. The effect of THP-1 monocytes EVs exert on endothelial cell physiological function Meanwhile, Western blotting was also used to examine some endothelial function associated proteins followed treated with monocytes EVs. Such as eNOS(regulates vascular tone and angiogenesis), Cyclin D1(a protein required for the G1 phase of the cell cycle), BAX and P53(important factors to the apoptosis). However, all those proteins show no significant change or statistical significance between basal with treated groups (see Fig. 3).
3.4. The effect of monocytes EVs exert on endothelial cell migration Through statistical analysis of the ratio of cell-free area to premier scratch area, we found that PA-EVs treatment enhances the migration function of endothelial cell greatly(As Fig. 4A/B shown). Accordantly, this pro-migration ability of PA-EVs get further confirmation in transwell assays (See Fig. 4C/D). In addition, in both scratch and transwell assay, the difference among the result of the four groups shows the maximal value in 12 h, while the value decreased in 24 h, reveals only a minor change in cell number. The major part of the cause is that, in the scratch assay, contact inhibition happened along with the migration of
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endothelial cells. In transwell assays, major cells migrated to the underside in 24 h, which weaken the difference between the groups. Taken together, these results suggest that palmitate-treated monocytes EVs may play a promoter role in the migration capacity of endothelial cells, whereas EVs participate in abnormal lipid metabolism associated complications.
4. Discuss As a hallmark of obesity, elevated plasma concentrations of FFA also has long been observed frequently in patients suffering from vascular disease [28]. Both animal and in vitro experience reveals saturated fatty acid triggers multiple pathological effects on cell types involved in atherogenesis. Palmitate stimulation also amplifies inflammation via Toll-like receptors [29] and induce IL-6 expression [30] in human monocytes. Whereas, the mono- and polyunsaturated fatty acids expressed few pro-inflammation effect [31]. The cargo of EVs varies with the physiological status of source cells, which endows EVs with multiple functions, as the role of specific “first messenger”. Although the direct effects of SFAs on inflammatory responses in endothelial cells [32,33] have been investigated, it remains
Fig. 3. The effect of THP-1 monocytes EVs exert on endothelial cell physiological function Representative Western blots showing the ratio of (A) eNOS, (B) Cyclin D1, (C) BAX, (D) p53 Basal:untreated HUVECs.NC,PA,SC:HUVECs treated with THP-1 monocytes stimulated by nothing, palmitate or solvent, respectively.
Please cite this article as: W. Gao et al., Monocyte-derived extracellular vesicles upon treated by palmitate promote endothelial migration and monocytes attachment to endothelial cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.095
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Fig. 4. The effect of monocytes EVs exert on endothelial cell migration (A) Scratch wound healing assay, take photos at 0,6,12,24 h respectively. Scratch wound distance is given by black lines. (B) Ratio of migration area to initial cell-free Scratch area. (C) Transwell migration assay was performed to determine the migration of HUVEC cells. (D) The column showed the quantification of the migrated cells in indicated groups in Transwell assays. All data values represent the mean ± SEM of triple independent experience *p < 0.05.
unknown whether FFA also induces similar responses mediated by EVs. In this experience, we found that saturated FFA (palmitate) change the proportion of monocytes EVs, in vitro, palmitate stimulated THP-1 monocytes EVs enhance the production of adhesion associated proteins, such as VCAM-1, ICAM-1. In Transwell and Scratch wound healing assay, EVs also exhibit pro-migration function on endothelial cells. In conclusion, we show a new pathological junction between FFA and endothelial damage that monocytes EVs participated in. our findings could provide a new sight of how complex interactions between obesity and vascular complication, which not only affect future research but also identify new targets for blood lipid control.
Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements This work is supported by the Department of Science and Technology, Henan Province (grant number: 122102310620).
Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.12.095.
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Please cite this article as: W. Gao et al., Monocyte-derived extracellular vesicles upon treated by palmitate promote endothelial migration and monocytes attachment to endothelial cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.095
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Monocyte-derived extracellular vesicles upon treated by palmitate promote endothelial migration and monocytes attachment to endothelial cells Wanhao Gao 1, Xingchen Guo 1, Ying Wang, Dongdong Jian, Muwei Li* Department of Cardiology, People’s Hospital of Zhengzhou University, Zhengzhou University, Zhengzhou, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 December 2019 Accepted 28 December 2019 Available online xxx
Aim: High circulating free fatty acid (FFA) concentration has a critical role in the development of obesity associated vascular comorbidities. Ample previous findings revealed that FFA, especially saturated, induce endothelial dysfunction throught multiple mechanisms (summarized as lipotoxicity). As a mediator that transfers information among cells, extracellular vesicles(EVs) participate in pathologic processes of many diseases, including angiocardiopathy, insulin resistance, autoimmunity disease. However, how lipotoxicity changed the proportion of EVs secreted from monocytes, furthermore, the effect of the EVs exerts on endothelial cells, haven’t been demonstrated. Method: In our experience, differential ultracentrifugation was used to extract EVs from condition medium (CM) of THP-1 monocytes under given treatments. Then we co-incubated the EVs derived from palmitate-treated monocytes with HUVECs for 24 h, after which molecular and phenotypic assays were conducted. Result: Palmitate-treated monocytes EVs promote the production of adhesion associated proteins of endothelial cells, such as VCAM-1, ICAM-1. Meanwhile, palmitate-stimulation may play a promoter role in the pro-migration capacity of monocytes-EVs. In brief, EVs could be the new pathological junction between FFA and endothelial damage. © 2020 Elsevier Inc. All rights reserved.
Keywords: Extracellular vesicles Monocytes Obesity Saturated FFA Endothelial cells
1. Introduction Around the beginning of the twenty-first century, obesity has emerged as an enormous public health problem globally [1]. These patients who confront an increased risk to develop vascular diseases, especially, atherosclerosis [2]. One of the characteristic abnormalities in obesity is high circulating FFA concentration. Clinical studies and in vivo studies have shown that high FFA levels are associated with impairment of endothelium-dependent vasodilation [3], insulin resistance [4,5], myocardial infarction [6], stroke [7]. In addition, FFA affect gene expression of Vascular Smooth Muscle Cells [8], adipocytes [9], macrophages [10,11] and monocytes [12]. Although FFA was
* Corresponding author. E-mail addresses: [email protected], [email protected] (W. Gao), [email protected] (X. Guo), [email protected] (Y. Wang), 11418149@ zju.edu.cn (D. Jian), [email protected] (M. Li). 1 These authors contributed equally to this work.
considered as one of the pathological junctions between obesity and endothelial damage, mechanisms of the FFA-induced comorbidities are still incompletely understood. Extracellular vesicles(EVs), the membrane-bound structure of cells [13], can be broadly classified into 3 main classes: Exosomes(released upon the fusion of multi-vesicular bodies), Microvesicles(outward budding and fission of plasma membrane), Apoptotic bodies(released as blebs of apoptotic cells). Although first discovered in the late 1960s, EVs were stated as an excretion mechanism through which cells eliminate unnecessary cellular metabolic wastes. Until 2007, a finding [14] pointed out the ability of EVs exchange cargo(containing proteins, mRNAs, and microRNAs) from donor cells to acceptor cells and triggering phenotypic changes in the latter, interest of the scientific community was attracted substantially. However, current EVs purify protocols are difficult to obtain pure EVs subtype [15], which brings a challenge to EVs research. Therefore, according to physical properties, EVs were simply classified as sEV (small EV) and lEV (large EV) in various studies. MISEV [16] also suggests that the EVs without strict origin demonstration, shouldn’t be
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Please cite this article as: W. Gao et al., Monocyte-derived extracellular vesicles upon treated by palmitate promote endothelial migration and monocytes attachment to endothelial cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.095
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named as exosomes or microvesicles, but distinguished by their size. EVs carry out their physiological functions in many pathophysiological situations, including immune responses [17],cardiovascular diseases [18] and cell-based therapy [19]. The recent finding also suggests [20], EVs play a significant role of mediator in endothelial cell function. Like most other cells, monocytes also release functional EVs [21]. Furthermore, monocyte-derived EVs diffuse in circulating blood, which greatly increased the possibility of uptaken by vascular endothelial cells. The relative proportion of released EVs is highly variable, which indicates the broader function EVs may possess. Therefore, we hypothesize that lipotoxicity induced monocytes EVs deliver molecules to endothelial cells, and participate in the development of endothelial dysfunction in obesity. 2. Material and methods 2.1. Antibodies and reagents Primary antibodies used for western blot included VCAM-1(Cat. No. ab134047), ICAM-1(Cat. No. ab109361), e-NOS(Cat. No. ab199956), p53(Cat. No. ab32389), Cyclin-D1(Cat. No. ab226977), TSG101(Cat. No. ab125011), Antibody HSP70(Cat. No. WL01019),Calnexin(Cat. No. WL03062) was purchased from Wanleibio(Shenyang, China), Antibody BAX (Cat. No. CY5059) was purchased from Abway(Beijing, China). E-selectin(Cat. No. 208941-AP), p65(Cat. No. 10745-1-AP) GAPDH(Cat. No. 10494-1-AP), beta-Actin(Cat. No. 20536-1-AP) were purchased from Proteintech Group (Proteintech, USA). Endothelial cell medium(ECM) was purchased from Sciencell(CA, USA); RPMI1640 and Fetal bovine serum (FBS) were purchased from Invitrogen(Carlsbad, USA). Saturated FFA palmitate (16: 0) and its solvent(Cat. No. SYSJ001) were purchased from Kunchuang biotechnology. RIPA buffer, SDS-PAGE Sample Loading Buffer, HRP-labeled Goat Anti-Rabbit/Mouse IgG, Coomassie Blue Staining Solution, Crystal Violet Staining Solution were purchased from Beyotime(Beijing, China). CFDA was purchased Solarbio(Beijing, China). Pierce BCA Protein Assay Kit was purchased from Thermofisher (Rockford, USA).
based on ultrafiltration to deplete EVs from FBS. Briefly, Ultra-15 Centrifugal Filter Devices (UFC910024, 100 kDa) were purchased from Merk Millipore(Darmstadt, Germany). EV-depleted FBS was prepared according to the studies of Kornilov et al. [22]. EVdepleted RPMI1640 medium was compound with 10% EVdepleted FBS. EVs isolation from monocytes CM: EVs were isolated and purified by classic differential ultracentrifugation [23], but with minor modifications. All the centrifugal steps described blew were conducted at 4 C. The CM of THP-1 monocytes was centrifuged at 600 g for 5 min to remove whole cells. And then centrifuged at 1200 g for 15 min to remove cell debris and apoptotic bodies. The resulting supernatant was centrifuged at 10,000 g for 20 min to remove lEVs. Finally, the supernatant was centrifuged in a Swinging-Bucket angle rotor (SW 32Ti, Beckman) at 100,000 g for 90 min to pellet sEVs. The pellet was resuspended in PBS and centrifuged again at 100,000 g for another 90 min. The final pellet was suspended in 100 ml PBS, and EVs were quantified by BCA assay (Thermo Scientific). EVs samples derived from THP-1 monocytes treated by palmitate (labeled as PA-EVs), Solvent (labeled as Solvent Control, SCEVs) or nothing (labeled as Normal Control, NC-EVs), were used in the subsequent analyses. EVs preparations were aliquoted and stored at 80 C and subjected to a single freeze-thaw. 2.4. Characterization of EVs Nanoparticle tracking analysis (NTA): The EVs concentration and size distribution was analyzed by NTA after diluting EVs preparations with PBS to obtain the optimal detection concentration. Three 40s videos were recorded and the data was analyzed using NTA software 3.0. Transmission electron microscopy (TEM): EVs samples isolated by ultracentrifugation were prepared for TEM and images described previously [24]. Samples were viewed with transmission EM using HT7800 TEM system(Hitachi Company) operating at 80 kV. Images were taken with 3295 2569 px image size. 2.5. EV treatments
2.2. Cell culture and stimulation THP-1 cells, obtained from American Type Culture Collection (Manassas, USA), were cultured in RPMI 1640 medium supplemented with 10% FBS, 100U/ml penicillin and 100 mg/ml streptomycin (all from Invitrogen, USA). Adjust the density of cell suspension ranged from 2 105 to 1 106 cells/mL during cell expansion. Prior to cell stimulation, collected THP-1 monocytes by centrifugal separation were suspended in medium containing EVs-depleted FBS (protocol described below), Then, to treat THP1 cell with saturated FFA palmitate (16: 0; 200 mM), Solvent control(equal volume with palmitate) or nothing for 24 h. Collecting conditioned media (CM) of different groups for isolation of EVs. Primary human umbilical vein endothelial cells (HUVECs), obtained from Sciencell (CA, USA), were maintained in endothelial cell medium containing 5%FBS, 2 mM glutamine, 100U/ml penicillin, and 100 mg/ml streptomycin. All the cells were cultured at 37 C and 5% CO2 in a humidified incubator. 2.3. EVs isolation Preparation of EV-depleted FBS: in order to minimize the effect of FBS EVs on downstream analysis. We use a protocol
HUVECs were plated on twelve-well plates for a maximum of 6 passages. Upon 60% confluence, HUVECs were treated with NCEVs(NC-Group), PA-EVs(PA-Group), SC-EVs(SC-Group) for 24 h, EVs was add in ECM of each treated group at a final concentration of 20 mg/ml. HUVECs treated with the same volume of PBS were used as an experimental control(Basal-Group), after this, HUVECs was used for protein extraction and other phenotypic assays. 2.6. Western blot analysis Western blotting was performed as described previously [25], Cells and EVs preparations were lysed with RIPA buffer at 4 C for 30 min, protease inhibitors were added in RIPA buffer in advance. After centrifuged at 12,000 g for 10 min at 4 C, lysed was collected and quantified by the BCA method. Use RIPA buffer to leveling the protein concentration. Protein preparations were denatured at 95 C for 10min in sample Loading Buffer. Equal solubilized proteins were added to polyacrylamide SDS gels. After transferred proteins onto polyvinylidene difluoride membranes (Millipore, USA), Membranes were incubated with Western Blocking Buffer for 60 min at room temperature and then soaked in indicated antibodies overnight at 4 C. After incubation, blots were washed three times in TBS-T for 10 min at RT. Blotting with a horseradish peroxidase-conjugated secondary antibody (diluted 1: 2000 in
Please cite this article as: W. Gao et al., Monocyte-derived extracellular vesicles upon treated by palmitate promote endothelial migration and monocytes attachment to endothelial cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.095
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TBST) for 1 h at room temperature and then visualizing by ECL Kit (Millipore, USA). quantification was performed using Image J software. 2.7. Monocyte attachment assay As described above,60%e80% confluent HUVECs were treated with PBS, NC-EVs, PA-EVs, SC-EVs in a 12-well plate, 20 mg/ml EVs was add in each treated group. 24 h later, the Attachment assay was conducted referring to an earlier report [26]. Briefly, removed the ECM in the wells and washed the residual medium by PBS twice times. THP-1 monocytes were stained by CFDA(Solarbio, China) for 30 min in advanced according to instruction. 1.0 ml suspension of the fluorescently-labeled THP-1 monocytes was added in each well containing a confluent monolayer of HUVECs. Co-incubating THP-1 monocytes and HUVECs for 1 h, after which, non-adherent THP-1 monocytes were removed by three times PBS washes. Five images from the area around the center of each well were captured by an inverted fluorescence microscope. The percentage of total image area covered by THP1 cells was measured by Image J software. 2.8. Scratch wound healing assay Scratch assays were performed as described previously [20]. Upon 90e100% confluence, the monolayer of HUVECs in 12-well plates was scratched with a sterile pipette (200ml). Then low-
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serum ECM (1% EVs-depleted FBS) was added to the wells and cells were stimulated with PBS,NC-EVs(20 mg/ml),PA-EVs(20 mg/ ml),SC-EVs (20 mg/ml) for 24 h, which same with above description. Cells were photographed on the marked position at 0, 6 12 24 h. Use image J to measure and correlated the remaining cell-free area to the initial scratched area (in percent). 2.9. Transwell migration assay Transwell assays were performed using 8.0-mm-pore transwell chambers (Corning, USA) with polycarbonate filters. The trial grouping method was the same as the Scratch wound healing assay but in EVs-depleted 5%FBS medium. Subsequently, collected HUVECs by centrifugal separation was resuspended and counted. Then 200 ml of HUVECs suspension (about 1 105/ml in ECM containing 1% FBS) was added in the upper chamber, whereas the lower chamber contained ECM supplemented with 10% FBS. After 12 or 24 h, the medium in the upper chamber was discarded. And then, using a cotton swab to abrase non-migrating cells from the upper surface of the membrane. Cells on the underside were fixed with 4% paraformaldehyde for 20 min and then stained with crystal violet staining solution for 10 min. Finally, washing and removed resident crystal violet three times by PBS. Counting the number of stained Migrating cells from four randomly chosen high-power fields ( 100) with a phasecontrast microscope (Olympus, Japan). All assays were performed in three separate sets of experiments.
Fig. 1. Characteristic of Extracellular Vesicle derived from THP-1 monocytes. (A)transmission electron micrograph revealing the typical morphology and size of EVs. (B) Western blot reveals the presence of the exosomal protein markers TSG101, HSP70. Proteins of THP-1 monocytes used as control group, identified GAPDH and calnexin undetectable in EVs preparations. (C) Coomassie Brilliant Blue staining of extracted proteins of THP-1 monocytes and EVs. NTA profiles confirmed the size distribution of the EVs preparations that secreted from normal THP-1 cell (D) or THP-1 cell treated with solvent-control (F) or palmitate(E). . (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Please cite this article as: W. Gao et al., Monocyte-derived extracellular vesicles upon treated by palmitate promote endothelial migration and monocytes attachment to endothelial cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.095
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2.10. Statistical analyses
to 75kda and no significant difference among three EVs groups.
Data are presented as mean ± SEM. *p < 0.05, **p < 0.01 and ***p < 0.001 by one-way ANOVA. A p-value < 0.05 was determined to be statistically significant. GraphPad software was used to perform all analyses and to construct all graphs.
3.2. The effect of monocytes EVs exert on endothelial cell inflammation
3. Result 3.1. Characterization of EVs EVs from THP-1 monocytes CM were isolated by ultracentrifugation and visualized by TEM after negative staining. As shown in Fig. 1A, isolated EVs possess typical round, oval or cup-shaped, lipid bilayer membranous vesicular morphology. Apart from the ultrastructure of EVs, TEM also reveals a size in a range of 30e150 nm in diameter sketchily. Furthermore, NTA profiles confirmed the size distribution of the EVs preparations, that the peak analysis(concentration)of EVs secreted from normal THP-1 cell (146.5 ± 2.28 nm, Fig. 1D) and THP-1 cell treated with palmitate (138.6 ± 2.12 nm, Fig. 1F) was similar to that treated with solventcontrol(142.2 ± 0.96 nm, Fig. 1E). Upon further characterization by Western blot, using TSG101 and HSP70 as Exosomal marker, Calnexin as an endoplasmic reticulum marker, GAPDH as a cytoplast marker. In Fig. 1B, the blots of TSG101 and HSP70 proteins reveals distinct traces, while small amounts of Calnexin and GAPDH proteins could be detected, which confirms that the EVs preparations were free of contaminating proteins. Coomassie Brilliant Blue staining(Fig. 1C) suggesting molecular weight distribution of EVs proteins separated on SDS-PAGE gels, whose peaks(concentration)range from 60kda
Monocytes attached to activated human vascular endothelium is the initial step of atherogenesis. To detect the effect of monocytes derived EVs exert on HUVECs, monocytes attachment assays were implemented initially. Co-incubating Fluorescent dyed and numerically equal THP-1 cells with HUVECs, which pretreated with NC-EVs, PA-EVs, SC-EVs or PBS for 24 h. As Fig. 2Eshown, PA-EVs treat groups exhibit a significantly increased monocytes attachment, compared to those observed for HUVECs didn’t treated with EVs. The trendy was inexistent among the other three groups. To confirm it, following treatment of HUVECs with monocytes EVs, Western blotting was used to detect the production of adhesion associated proteins involved in monocytes attachment. As Fig. 2A/B shown, the production of the protein: ICAM-1 and ICAM-1 was increased in HUVECs treated with PA-EVs compared to that of untreated HUVECs(Basal Group). No detectable change in NC-EVs nor SC-EVs Group. The level of E-selectin protein has no significant change in both PA-EVs and NC-EVs groups. Although slightly increasing trend was observed in SC-EVs groups, there is no statistical significance was obtained (P ¼ 0.1681). As a crucial regulation protein of inflammation signal, NF-ΚB also participates in the pro-inflammation function of EVs, as research reported earlier [27]. However, in our experiment, lipotoxicity induced monocytes derived EVs didn’t change the proteins level of p65 in HUVECS.
Fig. 2. The effect of monocytes EVs exert on endothelial cell inflammation Representative graph showing the ratio of (A) ICAM-1, (B) VCAM-1, (C) E-Selectin, (D) p65 expression in HUVECs following treatment with PBS(Basal), NC-EVs(NC), PA-EVs(PA), SC-EVs(SC). Representative blots of proteins are shown in Fig. 2G. (E)Monocytes-endothelial cells attachment assay and (F) Ratio of percentage area covered by monocytes to a confluent monolayer of HUVECs. Green dots are THP-1 monocytes stained with CFDA All data values represent the mean ± SEM of triple.independent experience *p < 0.05, **p < 0.01. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Please cite this article as: W. Gao et al., Monocyte-derived extracellular vesicles upon treated by palmitate promote endothelial migration and monocytes attachment to endothelial cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.095
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3.3. The effect of THP-1 monocytes EVs exert on endothelial cell physiological function Meanwhile, Western blotting was also used to examine some endothelial function associated proteins followed treated with monocytes EVs. Such as eNOS(regulates vascular tone and angiogenesis), Cyclin D1(a protein required for the G1 phase of the cell cycle), BAX and P53(important factors to the apoptosis). However, all those proteins show no significant change or statistical significance between basal with treated groups (see Fig. 3).
3.4. The effect of monocytes EVs exert on endothelial cell migration Through statistical analysis of the ratio of cell-free area to premier scratch area, we found that PA-EVs treatment enhances the migration function of endothelial cell greatly(As Fig. 4A/B shown). Accordantly, this pro-migration ability of PA-EVs get further confirmation in transwell assays (See Fig. 4C/D). In addition, in both scratch and transwell assay, the difference among the result of the four groups shows the maximal value in 12 h, while the value decreased in 24 h, reveals only a minor change in cell number. The major part of the cause is that, in the scratch assay, contact inhibition happened along with the migration of
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endothelial cells. In transwell assays, major cells migrated to the underside in 24 h, which weaken the difference between the groups. Taken together, these results suggest that palmitate-treated monocytes EVs may play a promoter role in the migration capacity of endothelial cells, whereas EVs participate in abnormal lipid metabolism associated complications.
4. Discuss As a hallmark of obesity, elevated plasma concentrations of FFA also has long been observed frequently in patients suffering from vascular disease [28]. Both animal and in vitro experience reveals saturated fatty acid triggers multiple pathological effects on cell types involved in atherogenesis. Palmitate stimulation also amplifies inflammation via Toll-like receptors [29] and induce IL-6 expression [30] in human monocytes. Whereas, the mono- and polyunsaturated fatty acids expressed few pro-inflammation effect [31]. The cargo of EVs varies with the physiological status of source cells, which endows EVs with multiple functions, as the role of specific “first messenger”. Although the direct effects of SFAs on inflammatory responses in endothelial cells [32,33] have been investigated, it remains
Fig. 3. The effect of THP-1 monocytes EVs exert on endothelial cell physiological function Representative Western blots showing the ratio of (A) eNOS, (B) Cyclin D1, (C) BAX, (D) p53 Basal:untreated HUVECs.NC,PA,SC:HUVECs treated with THP-1 monocytes stimulated by nothing, palmitate or solvent, respectively.
Please cite this article as: W. Gao et al., Monocyte-derived extracellular vesicles upon treated by palmitate promote endothelial migration and monocytes attachment to endothelial cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.095
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Fig. 4. The effect of monocytes EVs exert on endothelial cell migration (A) Scratch wound healing assay, take photos at 0,6,12,24 h respectively. Scratch wound distance is given by black lines. (B) Ratio of migration area to initial cell-free Scratch area. (C) Transwell migration assay was performed to determine the migration of HUVEC cells. (D) The column showed the quantification of the migrated cells in indicated groups in Transwell assays. All data values represent the mean ± SEM of triple independent experience *p < 0.05.
unknown whether FFA also induces similar responses mediated by EVs. In this experience, we found that saturated FFA (palmitate) change the proportion of monocytes EVs, in vitro, palmitate stimulated THP-1 monocytes EVs enhance the production of adhesion associated proteins, such as VCAM-1, ICAM-1. In Transwell and Scratch wound healing assay, EVs also exhibit pro-migration function on endothelial cells. In conclusion, we show a new pathological junction between FFA and endothelial damage that monocytes EVs participated in. our findings could provide a new sight of how complex interactions between obesity and vascular complication, which not only affect future research but also identify new targets for blood lipid control.
Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements This work is supported by the Department of Science and Technology, Henan Province (grant number: 122102310620).
Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.12.095.
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Please cite this article as: W. Gao et al., Monocyte-derived extracellular vesicles upon treated by palmitate promote endothelial migration and monocytes attachment to endothelial cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.095