Iron oxide nanoparticles induce reversible endothelial-to-mesenchymal transition in vascular endothelial cells at acutely non-cytotoxic concentrations
Iron oxide nanoparticles (IONPs) have been extensively studied in different biomedical fields. Recently, the non-cytotoxic concentration of IONPs induced cell-specific response raised concern of their safety. Endothelial cell exposure was unavoidable in their applications, while whether IONPs affect the phenotype of vascular endothelial cells is largely unknown. In this work, the effect of IONPs on endothelial-to-mesenchymal transition (EndMT) was investigated in vitro and in vivo.
The incubation with γ-Fe2O3 nanoparticles modified with polyglucose sorbitol carboxymethyether (PSC-Fe2O3) at non-cytotoxic concentration induced morphological changes of human umbilical vein endothelial cells (HUVECs) from cobblestone-like to spindle mesenchymal-like morphology, while PSC-Fe2O3 mostly stay in the culture medium and intercellular space. At the same time, the endothelial marker CD31 and VE-cadherin was decreased along with the inhibitory of angiogenesis properties of HUVEC. Meanwhile, the mesenchymal marker α-smooth muscle actin (α-SMA) and fibroblast specific protein (FSP) was up regulated significantly, and the migration ability of the cells was enhanced. When ROS scavenger mannitol or AA was supplemented, the EndMT was rescued. Results from the in vivo study showed that, expression of CD31 was decreased and α-SMA increased in the liver, spleen and kidney of mice given PSC-Fe2O3, and the density of collagen fibers in the liver sinusoid of mice was increased. The supplementary mannitol or AA could reverse the degree of EndMT in the tissues. Mechanistic study in vitro indicated that the level of extracellular hydroxyl radicals (·OH) was up regulated significantly by PSC-Fe2O3, which induced the response of intracellular ROS and resulted in the EndMT effect on HUVECs.
The PSC-Fe2O3 was capable of inducing EndMT in the endothelial cells at acutely non-cytotoxic dose due to its intrinsic peroxidase-like activity, though they were few taken up by endothelial cell. The EndMT effect on HUVEC can be rescued by ROS scavenger in vitro and in vivo.
KeywordsIron oxide nanoparticle Endothelial-to-mesenchymal transition Reactive oxygen species Reversible
2′, 7′-dichlorodihydrofluorescein diacetate
5, 5-Dimethyl-1-pyrroline N-oxide
Electron spin resonance
Fibroblast specific protein
Human umbilical vein endothelial cells
Iron oxide nanoparticles
γ-Fe2O3 nanoparticle core and polyglucose sorbitol carboxymethyether shell
Reactive oxygen species
Transmission electron microscope
α-smooth muscle actin
Iron oxide nanoparticles (IONPs) have been intensively investigated and developed in many biomedical fields, such as intravenous cell targeting, labeling and separation, magnetic resonance imaging contrast, drug or gene delivery system, and hyperthermia [1, 2, 3], because they show a low cytotoxicity in a quite large range of tested concentration [4, 5]. Nevertheless, potential effects of IONPs at non-cytotoxic concentrations on human health have been explored in recent years, especially on vascular endothelial cells, because blood vessel is one of the major barriers for INOPs intended to use as therapeutics and diagnostics. For examples, it was reported that IONPs exposure induced the elongated morphology and increased elastic modulus of endothelial cells [6, 7]. Even no cytotoxicity was detected, at low amount of IONPs, the endocrine and urea transporter function, inflammatory responses, angiogenesis function of endothelial cells (ECs) were found to change . The endothelial integrity was also reported to alter via interplay with barrier function and attenuation of cytoprotective and anti-inflammatory NO production at non-cytotoxic concentration of IONPs . These cues imply possible effects of IONPs at non-cytotoxic concentration on the phenotype of ECs.
Endothelium to mesenchymal transition (EndMT) is one changed phenotype of endothelial cells that represents losing endothelial characteristics and gaining a mesenchymal phenotype , which have a significant role in some diseases, especially fibrosis in liver [11, 12], kidney , cardiac , and atherosclerosis , diabetes  and cancer . It also has been documented that endothelial cells can undergo EndMT in certain physiological environmental stimulations including growth factor TGFβ , materials stiffness , and stretching stress . Whether IONPs can induce EndMT is unknown and worthy to note.
In this work, whether γ-Fe2O3 nanoparticles modified with polyglucose sorbitol carboxymethyether (PSC-Fe2O3) were able to inducing EndMT upon human umbilical vein endothelial cells (HUVECs) was investigated in vitro and in vivo. We showed that PSC-Fe2O3 at acutely non-cytotoxic concentration significantly reduced the expression of endothelial marker CD31 and VE-cadherin, meanwhile increased the mesenchymal marker α-smooth muscle actin (α-SMA) and fibroblast specific protein (FSP) due to the peroxidase-like activity, by which the migration of endothelial cells was enhanced and their angiogenic function was inhibited, clearly indicating the occurrence of EndMT in the endothelial cells. The underlying mechanism of PSC-Fe2O3 induced EndMT was also elucidated.
Characterization and cytotoxicity of PSC-Fe2O3
PSC-Fe2O3 induce EndMT
PSC-Fe2O3 mainly stayed in the culture medium and intercellular space
Extracellularly PSC-Fe2O3 induced ROS increase by peroxidase-like activities
ROS scavengers reverse EndMT of HUVEC
ROS scavengers rescued PSC-Fe2O3 induced EndMT in vivo
Endothelial-to-mesenchymal transition (EndMT) is accompanied with a progressive loss of endothelial markers and cell-to-cell adhesion, transition towards a mesenchymal phenotype, and enhancement on cell migration and invasion, which has been recognized to play a diverse role in many diseases, such as fibrosis, systemic sclerosis, diabetes and malignancy . In this work, we revealed that PSC-Fe2O3 could induce EndMT at acutely non-cytotoxic concentration. In order to figure out whether the occurrence of EndMT came from the modification of PSC, we also performed experiments with bare Fe2O3 nanoparticles (bare-Fe2O3) of 10 nm in diameter (Additional file 1: Fig. S4A). The bare-Fe2O3 showed 50% of viability when the concentration was reached 300 μg/mL (Additional file 1: Figure S4B), and induced significant morphological changes of HUVECs, cells shape changed from polygonal to spindle at different concentrations less than 300 μg/mL (Additional file 1: Figure S4C). Different from the cellular uptake of PSC-Fe2O3, the bare-Fe2O3 was able to be engulfed by HUVECs evidenced by the increasing blue color in the cells. In further, western blot analysis showed that α-SMA was increased remarkably and VE-cad decreased at the same time in dose-dependent manner (Additional file 1: Figure S4D). These results indicated that bare-Fe2O3 was capable of inducing EndMT on HUVECs as well, suggesting that the induction of EndMT was closely related with the intrinsic property of NPs instead of surface modification and cellular uptake. Note, both PSC-Fe2O3 and bare-Fe2O3 were household, which should not be taken as equal ones to commercial products in application, considering that they may have different physicochemical properties. It was known that EndMT is a subtype of epithelial-mesenchymal transition (EMT). Several nanoparticles or nanostructures have been demonstrated to be able to induce EMT with epithelial cell in the pulmonary system or cancer cell, such as carbon nanotube [35, 36, 37, 38], cerium oxide , titanium dioxide (TiO2) , silica (SiO2) , silver NPs , which is supportive to our observations on PSC-Fe2O3-induced EndMT. These insights about PSC-Fe2O3-induced EndMT can also provide a new view to evaluate the potential health risk of this nanoparticle in further applications.
IONPs are able to induce the production of intracellular ROS because they are internalized by cells and located in the lysosomes where the microenvironment is acidic . Due to the surface modification, PSC-Fe2O3 were not engulfed by HUVECs, instead, they mainly adhered to the cell membrane or stayed in the intercellular space. Nevertheless, PSC-Fe2O3 were still capable of up-regulating the level of intracellular ROS by catalyzing extracellular H2O2 into ·OH that further induced oxidative stress of the cells and affected intracellular environment. Therefore we suggested that the intrinsic peroxidase-like activity of PSC-Fe2O3 brought out the EndMT effect. According to the mechanism, it was reasonable to consider that ROS scavengers could reverse the effect, which was validated by this study in vitro and in vivo. It was also important and encouraging to see that the supplementary of mannitol or AA could reduce the degree of EndMT, which implied that the combination of antioxidants with PSC-Fe2O3 was a promising way of prevention the risk of EndMT related toxicology for the pre- and clinical application with PSC-Fe2O3. Moreover, it was noticed that iron oxide nanoparticles could reduce the adherens conjugation between HUVECs and led to endothelial leakiness through increasing the production of ROS , which might be one of the potential mechanisms that trigger the occurrence of EndMT mediated by PSC-Fe2O3.
The peroxidase-like activity of PSC-Fe2O3 resulted in the up-regulation of intracellular ROS though they were mainly stay in the culture medium and intercellular space, which induced EndMT on HUVECs with the decreased level of CD31 and VE-cadherin, and increased expression of α-SMA and FSP. ROS scavenger mannitol or AA could rescue the EndMT induced by PSC-Fe2O3 in vitro and in vivo. These results provided a new insight into the potential toxicity of PSC-Fe2O3 on vascular endothelial cells and demonstrated ROS scavenge was an effective strategy to reverse the process and reduce the potential toxicity.
PSC-Fe2O3 synthesis and characterization
The polydextrose sorbitol carboxymethyl ether (PSC) coated γ-Fe2O3 nanoparticles (PSC-Fe2O3) were synthetized by alternating-current magnetic field inducing method . Bare Fe2O3 nanoparticles were prepared similar to PSC-Fe2O3 in the absent of PSC. The morphology of Fe2O3 was observed by transmission electron microscope (TEM-1400plus, JEOL). The hydrodynamic diameters and Zeta potential of PSC-Fe2O3 were detected by a Zetasizer Nano ZS90 analyzer (Malvern). Each measurement was repeated four times.
Human umbilical vein endothelial cells (HUVECs, #8000), endothelial cell medium (ECM, #1001), fetal bovine serum (FBS, #0025), penicillin/streptomycin solution (P/S, #0503), endothelial cell growth supplement (ECGS,#1052) and Poly-L-Lysine (PLL, #0403) were all purchased from ScienCell Research Laboratories (San Diego, CA). HUVECs were grown on PLL coated culture plate in ECM supplemented with 5% FBS, 1% P/S and 1% ECGS.
Cell viability assay
The viability of HUVECs incubated with Fe2O3 was analyzed using a cell count kit (CCK-8, Dojindo). The Fe2O3 was diluted in fresh medium to yield a final iron concentration that ranged from 0 to 600 μg/mL. After 24 h or 48 h incubation, the cells were rinsed twice with PBS and incubated with medium containing 10 μL CCK-8 reagents for 2 h. The absorbance of the medium was measured at 450 nm using a microplate reader (BioTek Synergy4). All measurements were carried out in triplicate, and the cell viability was calculated with the protocol provided by the manufacture.
Cell morphology assay
HUVEC cells were exposed to 0, 300 and 600 μg/mL PSC-Fe2O3. After 48 h, the morphology of cells was observed by the microscope (Olympus IX71). The length and diameter of thirty cells in each treatment was measured by ImageJ software, and the ratio of length to diameter (L/D) was calculated.
Cells were incubated with 0, 150, 300 and 600 μg/mL Fe2O3 for 48 h at 37 °C. HUVECs were lysed in RIPA buffer and analyzed by western blot. Primary antibodies were as follows: anti-VE-cadherin (#2500S, Cell Signaling Technology, Danvers, MA), anti-CD31 (#3528, CST), anti-α-smooth muscle actin (#19245S, CST), anti-FSP (#13018, CST) and anti-GAPDH (sc-25778, Santa Cruz). Protein bands were quantified using ImageJ software. For the rescue experiment, HUVECs were co-cultured by Fe2O3 with 0.88 mg/mL mannitol or 8.8 mg/mL ascorbic acid (AA).
Cells were treated with different concentrations of PSC-Fe2O3 for 48 h and then fixed with 4% paraformaldehyde for 15 min. After rinsing with PBS, cells were incubated with blocking solution (1% bovine serum albumin (BSA) in PBS with 0.3% Triton) for 1 h at room temperature. The following primary antibodies were applied overnight at 4 °C: anti-VE-cadherin, anti-CD31, anti-α-smooth muscle actin, and anti-FSP. Cells were washed with PBS, and corresponding fluorescent secondary antibodies (CST) were applied for 1 h at room temperature. Nuclear staining was performed use 4′,6-diamidino-2-phenylindole (DAPI, Zhongshan Goldenbridge). Micrographs were taken by the FluoView FV1000 confocal microscope (Olympus), and analyzed with ImageJ software. For the rescue experiment, the cells were treated with 600 μg/mL PSC-Fe2O3 and 0.88 mg/mL mannitol or 8.8 mg/mL AA. The paraffin-embedded liver, spleen, and renal sections were deparaffinized with xylene. After treatment with 3% BSA for 30 min, sections were incubated overnight with primary CD31 and α-SMA antibody. Slides were then washed and incubated in the dark for 50 min with corresponding alexa fluor® 488 or CY3 labeled secondary antibody. Slides were then again washed in PBS and counterstained with DAPI-containing mounting medium.
Cells were pretreated with 600 μg/mL PSC-Fe2O3 in the absent or present of 50 mM AA for 48 h before seeded in matrigel coated 96-well plates. Cells were allowed to attach for 8 h, stained with Calcien AM (Molecular Probe) and photographed by the fluorescence microscope (Olympus, IX71). Numbers of tubules were quantified using ImageJ with the Angiogenesis Analyzer plugin. Data presented were from three independent experiments.
Cells were pretreated with 600 μg/mL PSC-Fe2O3 with or without 50 mM AA for 48 h and then starved without FBS for 12 h. Wounds were made with 200 μL sterile pipette tip and washed with PBS to eliminate non adherent cells, thus a wound approximate 165 μm was created. Then cells were cultured with medium with 0.5% FBS and images were acquired at 0 and 12 h (Olympus IX71). Movements of cells were calculated using ImageJ software. The changes of scratch width were calculated with initial wound width subtracted to that of 12 h and then divided by the initial one.
Prussian blue staining
Cells were treated with different concentrations of Fe2O3 for 48 h. After removing the culture medium, the cells were washed with PBS for three times and fixed with 4% paraformaldehyde for 30 min. The fixed cells were incubated with Prussian blue solution containing 5% hydrochloric acid aqueous solution and 5% potassium ferrocyanide (II) trihydrate for 30 min to determine the intracellular iron followed by staining with nuclear fast red. The cells were placed on an inverted microscope for observation.
Transmission electron microscopy (TEM)
Cells were treated with 600 μg/mL PSC-Fe2O3 for 48 h and then detached with trypsin and fixed overnight with 2.5% glutaraldehyde at 4 °C. The samples were then postfixed in Osmium (VIII) tetroxide (OsO4), dehydrated in ethanol, and embedded in Epon (Fluka). Sections were cut with an ultra-microtome and placed on copper grids for observation (TEM-1400 plus, JEOL).
Iron content measurements
Cells were treated with PSC-Fe2O3 for 48 h. There are two different procedures to deal with HUVECs before detecting the iron content in cells. Procedure I: the cells were washed with PBS and lysed by 100 μL RIPA. Procedure II: the cells washed with PBS were detached from the plates with trypsin and collected by centrifugation, and then washed with PBS and lysed by 100 μL RIPA. The amount of iron was quantified with phenanthroline spectrophotometric method . The iron content in the culture medium was determined with the same method. Standard calibration of iron amount and absorbance at 510 nm was built by using series concentration (0, 20, 40, 60, 80, 100 μg/mL) of FeCl3 solution. The concentrations of iron in the lysates and medium were calculated from the standard curve. Data presented were from three independent experiments performed in triplicate.
Intracellular reactive oxygen species (ROS) measurement
The intracellular ROS level was measured using the probe 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA, Sigma-Aldrich). HUVECs were treated with various concentration of PSC-Fe2O3 for 24 h and the culture supernatant was collected. The cells were then washed with PBS for three times. Cells were incubated with 10 μM DCFH-DA for 30 min and then digested and collected. The fluorescence intensity was detected by flow cytometer (BD Accuri C6). For the rescue experiment, the cells were treated by PSC-Fe2O3 with 0.88 mg/mL mannitol or 8.8 mg/mL AA. Data presented were from three independent experiments performed in triplicate.
Extracellular H2O2 and ROS measurement
The extracellular H2O2 and ROS level was measured using the above collected supernatants. In order to avoid interference from PSC-Fe2O3, ultrafiltration centrifugal filter (10 K, Amicon®) was used to separate PSC-Fe2O3 from supernatants. Centrifugal filter was performed following the manufacturer’s recommendations. In short, the supernatants were added to the device, followed by centrifugation. Then, the solution in the filter collection tube was collected.
The H2O2 concentration in the filtered solution was detected by hydrogen peroxide assay Kit (Beyotime Biotechnology). Standard calibration of H2O2 was built by using series concentration (1, 2, 5, 10, 20 μM) of H2O2 solution (Beijing Chemical Works) with absorbance at 560 nm. The concentration of H2O2 in the filtered solution was calculated with the standard curve. Data from three independent experiments were presented.
To explore the effect of PSC-Fe2O3 on extracellular ROS production, we used a modified assay with DCFH-DA probe . DCFH-DA in DMSO was hydrolyzed in NaOH aqueous solution for 30 min to yield DCFH intermediate. The obtained DCFH solution was neutralized with NaH2PO4 and shielded from light. The obtained filtered supernatants were mixed with DCFH solution. After 30-min incubation, the fluorescence was detected (BioTek Synergy4) and the excitation and emission wavelengths were set at 488 and 525 nm, respectively.
Sequences information of the gene primers
In vitro peroxidase-like activity assay
The peroxidase-like activity of PSC-Fe2O3 was evaluated in deionized water with the catalytic oxidation of o-phenylenediamine (OPD). The peroxidase-like activities of different concentrations (0, 25, 50, 60 μg/mL) PSC-Fe2O3 were employed with 45 μg/mL OPD and 370 mM H2O2. The progress was monitored with UV-vis-NIR spectrophotometer (PerkinElmer Lambda 950) with 1-min intervals at 440 nm. All operations were done at room temperature and in the dark.
Electron spin resonance (ESR)
Hydroxyl radicals (·OH) can be detected by ESR with the help of the spin trap 5,5-Dimethyl-1-pyrroline N-oxide (DMPO, Dojindo). The ESR measurements were carried out using a Magnettech ESR spectrometer (MS-5000) at ambient temperature. 50 μL aliquots of control or sample solutions were taken in quartz capillary tubes. Other settings were as follows: 2 G field modulation, 100 G scan range, and 10 mW microwave power.
Animal experiments were carried out in accordance with a protocol that was approved by the Institutional Animal Care and Use committee (Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, and Peking Union Medical College). Female Balb/c mice (average weight 20 g) were maintained at the institutional experimental animal center under specific pathogen-free conditions. The mice were fed with sterilized food and autoclaved tap water.
The administration procedure of mice
Intragastrical administration (100 μL)
intravenous injection (100 μL)
4 mg/mL PSC-Fe2O3
PSC-Fe2O3 + AA
40 mg/mL AA
4 mg/mL PSC-Fe2O3
PSC-Fe2O3 + mannitol
4 mg/mL PSC-Fe2O3 + 5.87 mg/mL mannitol
The mice were treated once a day for 7 days and then sacrificed. The tissues were collected and fixed in 4% parformaldehyde for 24 h and subsequently embedded in paraffin for histopathological and immunohistochemistry analysis.
Histopathological and immunohistochemistry analysis
The paraffin-embedded tissue sections were deparaffinized with xylene. Endogenous peroxidase was blocked with 0.3% H2O2 for 25 min. After treatment with 3% BSA for 30 min, sections were incubated overnight with primary CD31 and α-SMA antibody. Slides were then washed and incubated with a horse radish peroxidase-linked secondary antibody for 50 min at room temperature, followed by diaminobenzidine and counterstaining with Mayer’s hematoxylin.
The slides were stained with Masson’s trichrome for evaluation of interstitial collagen deposition. Collagen area and total tissue area were measured using ImageJ and the IHC Tool Box plugin. Collagen volume fraction (CVF) was calculated dividing collagen area by the total area. For each group, four replicates were counted and calculated.
The data are shown as mean ± standard deviation (SD) for all treatment groups. Statistical significance was ascertained through one way ANOVA with SPSS software (SPSS17.0).
Consent of publication
TW, JM, and HYX designed experiments and made data analysis and wrote the main manuscript. LFD conducted main experiments and prepared data for Figs. BC provided the PSC-Fe2O3 and related data. TW, JM, DDY and AYY took part in part of the experiments. TW, JM, JL, NG and HYX discussed part sections of the manuscript. All authors reviewed the manuscript. All authors read and approved the final manuscript.
The work was supported by the National Key R&D Program of China (2017YFA0205504), CAMS Innovation Fund for Medical Science (CIFMS 2016- I2M-3-004 and 2018-I2M-3-006), and the National Natural Science Foundation of China (31771005 and 81801771).
Ethics approval and consent to participate
Animal experiments were carried out in accordance with a protocol that was approved by the Institutional Animal Care and Use committee (Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, and Peking Union Medical College, China). Female Balb/c mice were maintained at the institutional experimental animal center under specific pathogen-free conditions. The mice were fed with sterilized food and autoclaved tap water.
The authors declare that they have no competing interests.
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