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Review article

Received: 18 February 2017, Accepted: 23 February 2017 Published online in Wiley Online Library: 6 April 2017

(wileyonlinelibrary.com) DOI 10.1002/jat.3470

The use of human umbilical vein endothelial
cells (HUVECs) as an in vitro model to assess the
toxicity of nanoparticles to endothelium:
a review
Yi Caoa,b* , Yu Gonga, Liangliang Liub, Yiwei Zhoua,b, Xin Fanga,b,
Cao Zhanga, Yining Lia and Juan Lia
ABSTRACT: With the rapid development of nanotechnologies, nanoparticles (NPs) are increasingly produced and used in many
commercial products, which could lead to the contact of human blood vessels with NPs. Thus, it is necessary to understand
the adverse effects of NPs to relevant cells lining human blood vessels, especially endothelial cells (ECs) that cover the lumen
of blood vessels. Human umbilical vein endothelial cells (HUVECs) are among one of the most popular models used for ECs
in vitro. In the present review, we discussed studies that have used HUVECs as a model to investigate the EC–NP interactions,
the toxic effects of NPs on ECs and the mechanisms. The results of these studies indicated that NPs could be internalized into
HUVECs by the endocytosis pathway as well as transported across HUVECs by exocytosis and paracellular pathways. Exposure
of HUVECs to NPs could induce cytotoxicity, genotoxicity, eNOS uncoupling and endothelial activation, which could be explained
by NP-induced oxidative stress, inflammatory response and dysfunction of organelles. In addition, some studies have also
evaluated the influences of microenvironment (e.g. the presence of proteins and excessive nutrients), the physiological and/or
pathological stimuli related to the diversity of ECs (e.g. shear stress, cyclic stretch and inflammatory stimuli), and the
physicochemical properties of NPs on the responses of ECs to NP exposure. In conclusion, it has been suggested that HUVECs
could be considered as a relatively reliable and simple in vitro model for ECs to predict and evaluate the toxicity of NPs to
endothelium. Copyright © 2017 John Wiley & Sons, Ltd.

Keywords: nanoparticles; human umbilical vein endothelial cells; cytotoxicity; genotoxicity; eNOS uncoupling; endothelial activation

Introduction suggested that the interactions between ECs and NPs should be
carefully assessed to better understand the potential in vivo effects
The rapid development of nanotechnologies has led to the of NPs (Setyawati et al., 2015). Second, ECs play a pivotal role in the
increased use of nanoparticles (NPs) in many commercially regulation of blood vessel tone, thrombogenicity, homeostasis,
available products. According to a recent survey, 1814 consumer monocytes recruitment, and hormone transport, and dysfunction
products from 622 companies in 32 countries contain at least of ECs has been implicated in the development of a number of
one type of NPs, which may result in the exposure of human cardiovascular diseases (CVD), e.g. atherosclerosis (Libby, 2012;
beings to particles via dermal, inhalational or oral contact (Vance Gimbrone and Garcia-Cardena, 2016). Thus, the assessment of
et al., 2015). Meanwhile, NPs may be potentially used in the toxicity of NPs to ECs on the key events associated with the
biomedicine for disease therapy and imaging (Lewis and Kannan, progression of CVD (Fig. 2) may provide important information
2014; Nguyen and Zhao, 2015; Cerqueira et al., 2015). All of these about the side effects of NPs to cardiovascular systems
applications of NPs may lead to the contact of human blood (Tomaszewski et al., 2015). During the early development of
vessels with NPs, and the effects of NPs on blood vessels have atherosclerosis, ECs are activated in response to pro-atherogenic
gained extensive attention. One simple way of judging this is to stimuli and over-express adhesion molecules, e.g. intercellular cell
search available publications concerning NPs and blood vessels adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule 1
in PubMed. As shown in Fig. 1, querying PubMed with the term
‘nanoparticles and blood vessels’ revealed an increase in
publications on this topic especially in the past 5 years. Although
the number of publications does not always equalize scientific *Correspondence to: Yi Cao, Key Laboratory of Environment-Friendly Chemistry and
Applications of Ministry Education, Laboratory of Biochemistry, College of
quality, it suggests that the effects of NPs on blood vessels
Chemistry, Xiangtan University, Xiangtan 411105, China.
represent an unresolved and attractive scientific topic. E-mail: caoyi39@xtu.edu.cn
The lumen of human blood vessels is covered by a thin layer of
a
endothelial cells (ECs). In nanotoxicological and/or nanomedicine Key Laboratory of Environment-Friendly Chemistry and Applications of Ministry
studies, ECs are of particular interests for two main reasons. First, Education, Laboratory of Biochemistry, College of Chemistry, Xiangtan University,
Xiangtan 411105, China
ECs serve as the first contact for NPs entering the blood before
NPs are delivered to targets. Therefore, although only some of b
Institute of Bast Fiber Crops, Chinese Academy of Agricultural Sciences, Changsha
the NPs are intended to target the blood vessels, it has been 410205, China
1359

J. Appl. Toxicol. 2017; 37: 1359–1369 Copyright © 2017 John Wiley & Sons, Ltd.

Y. Cao et al. E-selectin. is not fully known. HUVECs have been considered as a general model oxygen species (ROS) rather than NO in the context of CVD for ECs both in normal and diseased conditions.. 2014. The key events associated with the early development of cardiovascular diseases (CVD). Ltd. HUVECs express many 1998 to 2016. The factors that may influence the toxicological responses of HUVECs to NP exposure are also discussed. 37: 1359–1369 ... 2017). 2013.. Baudin et al.. HUVECs as a model for ECs The primary isolated HUVECs are probably the most popular ECs used in research because human umbilical veins are relatively more available than other types of blood vessels. One of the most popular permanent endothelial cell lines exposure to NPs could affect the cardiovascular system and is EA. proper conditions. there may still be fundamental differences of publications in PubMed (Fig.. to recruit monocytes (Libby. [Colour figure can be viewed at wileyonlinelibrary. 2016b. PECAM-1. Gimbrone and Garcia-Cardena.hy 926 cells were more in vitro models for ECs. Moreover. 2017. the immortalized endothelial cell line do NPs influence these key of CVD. especially the function of has also been developed by the fusion of HUVECs with permanent ECs. These key events include expression of adhesion molecules. the toxic effects and the molecular mechanisms. Monocytes adhere firmly spheroid cultures (Heiss et al.com/journal/jat Copyright © 2017 John Wiley & Sons. as well as signaling molecules associated with vascular physiology such as NO (Boerma et al. 2016). Tabas and stimuli such as high glucose.hy 926.. Toxicol.. 2016). as revealed by the accumulation et al. We first discuss the validation of HUVECs as an in vitro model for ECs. See more 1360 in the text.hy 926 cells.. Appl. particularly vWF. Caniuguir et al.. Then we discuss the interactions between NPs and HUVECs. Under (VCAM-1) and selectins. (Karbach et al. Thus. important endothelial markers. Cao et al. 2013) as advanced models to better excessive lipids and transform into macrophage foam cells. lipopolysaccharide (LPS) and shear Bornfeldt.. differentiate into macrophages. cancer cells (Edgell et al.. 2016). Human umbilical vein between HUVECs and EA. 2016). Andrejecsk et al. VCAM-1 and selectins. of nitric oxide (NO) owing to the uncoupling of endothelial NO although human umbilical veins are only at certain stages of synthase (eNOS uncoupling). we aimed at susceptible to oxidized low-density lipoprotein (ox-LDL) induced Figure 2. 2005.g. HUVECs could be isolated and maintained by standard protocol with a relatively minimal requirement (Marin et al.. 2013. especially when they are endothelial cells (HUVECs) are among one of the most used exposed to stimuli. e... 2010. Walshe et al. Accumulations of publications concerning ‘nanoparticles and blood vessel’ and ‘nanoparticles and endothelial cells’ in PubMed from purchased from commercial resources. How Besides the primary cells. EA. Jang et al. 2013. Boerma about the effects of NPs to ECs.. HUVECs could be differentiated into 3D Gimbrone and Garcia-Cardena. 2007) or Figure 1. there is a gradual loss of bioactivity stress (Patel et al. 2016). Flt-1 and KDR (Baranska et al. 2006.. ICAM- interactions. which was derived by fusing HUVECs with A549 lung accelerate the development of atherosclerosis (Donaldson et al. it is not surprising that there is a scientific concern 1. which in turn produces reactive human life. In addition.hy 926 cells retained most of the Given the importance of ECs in understanding NP–blood vessel important endothelial markers. For example.. transformation of monocytes into macrophages and endothelial NO synthase uncoupling (eNOS uncoupling). HUVECs have the accumulation of macrophage foam cells within the arterial wall been shown to be responsive to physiological and/or pathological is a hallmark of atherosclerosis (Moore et al. However. Analysis of the gene and protein 2013. We hope that this review may help future studies that consider using this model to assess the toxicity of NPs to ECs. Moller et al. monocyte adhesion. 2006). In the present review. 1983). 2015) or 3D co-cultures (Chowdhury onto the activated ECs. 1). VCAM-1. 2012.com] wileyonlinelibrary. profile indicated that EA. 2001. J. engulf et al. ICAM-1. and understand the behavior of ECs in vivo. but convincing data suggested that cell lines. providing a comprehensive review of the use of HUVECs as an in vitro model for the assessment of toxicity of NPs to ECs.

2015. dependent on actin remodeling. 3). Ltd. HUVECs showed a decreased cellular viability. whereas smaller particles could be internalized by clathrin-mediated (dependent on ligand-receptor interaction) or caveolin-mediated (lipid raft involved) endocytosis. 2015). 2017). For this reason. all of these studies indicated that Recently. thus the Clathrin-mediated endocytosis is dependent on the ligand- obstacle of limited supplies of ECs could be overcome... mechanisms for the internalization of NPs into HUVECs or other Despite these advantages.hy 926 cells. the primary cells should be considered mediated endocytosis (Fig. 2017. However. it is expected that the example. Nevertheless. Appl. stem cells (iPS) and further differentiated into ECs.. it is still NPs are intended to target a specific type of ECs (Setyawati et al. they can still 2015). Particles could also be transported across HUVECs by exocytosis or through paracellular pathway by tight junction (TJ) opening. For Adriani et al. (2013) showed that phagocytosis was activated in HUVECs et al.. HUVECs are not as phagocytic as macrophages.. if it is necessary to use immortalized EC cell lines There are four distinct endocytosis pathways. whereas this response is absent in HUVECs (Holthe et al. These examples indicated (an immortalized HUVEC cell line) via macropinocytosis and that the endothelial heterogeneity from different tissues should clathrin-mediated endocytosis (Solarska-Sciuk et al... 2014. 2016c. 2012).. It Moreover. Dittmar et al. possible that HUVECs internalize nanodiamonds through similar 2015). expression of all the major integrin receptors involved in angiogenesis (Baranska et al. although HUVECs have been co-cultured with engulf certain types of NPs through phagocytosis or astrocytes to mimic the BBB in vitro (Wilhelm and Krizbai. Therefore. 2015). Large particles could be internalized into HUVECs by phyagocytosis or macrophinocytosis. whereas Agarwal et al. the differences between HUVECs and ECs in nanomedicine and nanotoxicology studies (Setyawati ECs from other origins should be noted.. detonation nanodiamonds stress and an inflammatory response in HUVECs (Gong et al. The iPS. Thus.. pathways. The mechanisms for NP-HUVEC interactions. (2012) showed the internalization of interactions between NPs and HUVEC-astrocyte co-cultures could DNA-coated SWCNT (single-walled carbon nanotube) into HUVECs be different compared with the real BBB if the transport of NPs is via Rac1-mediated macropinocytosis.hy were similar (White et al.. therefore. For example. Similarly. Kafshgari et al. dependent on cytoskeleton remodeling to form and transferrin receptor (responsible for iron binding transferrin plasma membrane ruffles. 2015). Cheng macropinocytosis and clathrin-mediated pathways. which may be responsible for some of palmitic acid did not significantly affect NP-induced oxidative amount of NP uptake. that could be considered as the as the first choice as they may better resemble the ECs in vivo. 2012. whereas EA. and glycoprotein (responsible for drug transport from brain to blood) macropinocytosis. Wu et al. Phagocytosis.. 2014.com/journal/jat . Bhattacharya et al. In response to high glucose. HUVECs (Rombouts et al. the iPS-derived ECs 926 cells were more sensitive to radiation compared with that of could be used as a useful model for ECs as primary ECs. wileyonlinelibrary. Nevertheless.. Although brain barrier (BBB) whereas absent in HUVECs (Setyawati et al. Cao et al. 2014. 2005). Blechinger et al. which is associated with reduced use this model. Kafshgari et al.. 2013). be considered to study the NP–EC interactions especially if the Although this study did not use the primary HUVECs..or caveolin-mediated endocytosis Figure 3.. Another (2013) showed that polyethylene glycol diacrylate (PEGDA)-based example is that human microvascular endothelial cells could be nanodisc and nanorod were internalized into HUVECs both by activated in vitro by palmitic acid (Maloney et al.hy 926 Interactions between NPs and HUVECs cells exhibited increased growth. Toxicol. In another study.HUVECs as an in vitro model toxicity owing to relatively lower levels of antioxidant systems derived from human iPS cells and human embryonic stem cells (Claise et al. cautions should be used for these phagocytosis. 2005). See more in 1361 the text. are typically involved in the uptake of transport) are expressed consecutively in ECs lining the blood– large particles (Oh and Park. 2009. 2015). 2015). 37: 1359–1369 Copyright © 2017 John Wiley & Sons. clathrin-mediated and caveolin- differences. a recent study showed that EA. et al.. [Colour figure can be viewed at wileyonlinelibrary. it has been shown that somatic cells (for example.. derived ECs are of high purity and with the expression of whereas caveolin-mediated endocytosis involves invagination of endothelial markers (Margariti et al.. 2014). Liu et al.. 1999). macropinocytosis. mediated by p-glycoprotein and transferrin receptor. namely such as EA. it is not surprising that the presence after exposure to silica NPs.. macropinocytosis (Supplementary Information Table 1). particles by lipid raft (Oh and Park. 2013). phagocytosis and macropinocytosis pathways could be activated fibroblasts) could be reprogrammed into the induced pluripotent in HUVECs in response to certain types of NPs for the uptake. 2012). ( grain size <20 nm) were shown to be internalized into HUVEC-ST 2016. the analysis of gene expression indicated that ECs has been shown that clathrin. EA. p. but both of them showed similar Endocytosis of NPs by HUVECs oxidative stress and impaired NO bioavailability (Karbach et al.com] J. receptor interaction which triggers the formation of coated pits.hy 926 cells were significantly the experiences with stem cells may be required to generate and less responsive to VEGF.

The toxic effects of NPs to HUVECs and the possible mechanisms. 4) (Azhdarzadeh et al. because junctional adhesion molecule-1 ( JAM-1) and claudin-5 as well as these pathways involve ligand-receptor interactions or lipid raft disrupted the formation of zonula occludens-1 (ZO-1). In HUVECs. One advantage of these studies to target caveolae in et al. developed to reflect. apoptosis.. using HUVECs may not reflect the transport HUVECs is that NPs taken up by caveolin-mediated endocytosis of NPs to cross all types of endothelium. 37: 1359–1369 . whereas the uptake of CdTe junction proteins. (2013) constructed CGKRK. Oxidative stress. contrast. For example. genotoxicity (DNA damage). In another could modulate endothelial permeability through the regulation study. lysosomal stabilization. 3) (Zhang and Yang. (2011) showed that have been convincingly shown to be cytotoxic to HUVECs Figure 4. mitochondrial Exocytosis by HUVECs has been observed after exposure to viability.com/journal/jat Copyright © 2017 John Wiley & Sons. 2011). [Colour figure can be viewed at wileyonlinelibrary. but the (Fig. inflammation and dysfunction of organelles could be the mechanisms for 1362 the adverse effects of NPs to HUVECs. et al. (2012) showed that exposure of HUVECs to multi-walled with cyclic Asn-Gly-Arg (cNGR) peptide for gene delivery through carbon nanotube (MWCNT) was associated with decreased TEER the caveolin-mediated pathway with relatively low toxicity to (transendothelial electrical resistance) and expression of ZO-1. Moreover. 2015)..g. a number of assays have been regulation of junction proteins (Fig. it should be noticed that ECs from Softisan 100) modified with poly(styrene sulfonate) which were different origins express different kinds and levels of junction preferentially internalized by HUVECs via the caveolin-mediated proteins to maintain the endothelial barrier function (Setyawati pathway.. For clathrin-dependent endocytosis. apoptosis and LDH release).. Similarly. 2016). See more in text. 2014.. transport was minimal.. poly(lactic acid)-poly(ethylene glycol) (PLA–PEG) NPs modified Xu et al. Some types of NPs. Toxic effects of NPs to HUVECs Exocytosis and paracellular transport of NPs Cytotoxicity NPs may also transport across ECs to targets via exocytosis (Oh and Park. whereas the reverse Au NPs (Klingberg et al. (2014) synthesized non-toxic lipid NPs (based on of junction proteins. 2016. Toxicol.com] wileyonlinelibrary. 2016). 2017. Frohlich. Ltd. 2014). membrane integrity.. 2015). Li et al. Sun et al. Slowing et al. Similarly. Therefore. 2012b) and silicon QD (Ohta metal-based NPs that are partially soluble to release metal ions. the changes in targeted drug delivery and to reduce side effects (Supplementary TJ proteins were associated with increased paracellular Information Table 1). J. 3). 2015b) and SiO2 NPs (Blechinger et al. transported into HeLa cells via exocytosis.and the expression of typical tight junction (TJ) proteins occludin. caveolin-mediated pathways (Yan et al. Cao et al. and it is necessary to use can bypass the lysosome to escape lysosomal degradation (Voigt the proper model of ECs according to the target of NPs.. which indicated that exocytosis could be 2013) were shown to be internalized into HUVECs mainly through a possible way for NP transport from ECs to target cells. particular functionalized Au NPs (Bartczak et al. expression of adhesion molecules and monocyte adhesion). Ag NPs were shown to promote the permeability through functionalized PEG-co-PCL NPs that were taken up by lipid the disruption of AJ protein VE-cadherin rather than TJ proteins in raft/caveolae-mediated endocytosis with the involvement of HUVECs (Guo et al. 2012). Voigt et al. However. Liu et al. Y. All of these studies in microtubules in HUVECs and showed the ability of the NPs to combinations indicated that exposure of HUVECs to solid NPs deliver anti-cancer drugs to inhibit tumor angiogenesis. is the predominant pathway involved in the internalization of NPs mesoporous silica NPs internalized into HUVECs could be into HUVECs (Supplementary Information Table 1).. 2016) or paracellular pathways through the To indicate the cytotoxicity of NPs. (2011) formulated permeability in HUVEC monolayer and the BBB in mice. and cell proliferation mesoporous silica NPs (Slowing et al. Moreover. several studies modified NPs to enhance specific expression of adherens junction (AT) proteins VE-cadherin and β- internalization via particularly caveolin-mediated pathways for catenin was not significantly affected. Exposure to NPs may induce cytotoxicity (for example decreased mitochondrial viability. Moreover. (2015) showed that Au NPs decreased quantum dots (QD) was dependent on both clathrin.. peptide (Fig. Appl. 2016). eNOS uncoupling and endothelial activation (release of inflammatory mediators. For example.. et al. Hu et al. e.

Calarco et al. well as Cd-based QD. Suzuki et al.. 2014). Au NPs segments of mice. Su et al. Liu et al.. 2011. the expression of ICAM-1 and VCAM-1 was increased in HUVECs induced DNA damage was associated with G2/M cell cycle after exposure to CB NPs... but the release of IL-6. TiO2 and ZnO NPs was on polymers. metal-based NPs. Diminished NO increased lysosomal destabilization and apoptosis (Tsou et al.. Duan et al.. it has been shown that several types expression of adhesion molecules. 2012.. could produce NO through the activity of shown to induce cytotoxicity of HUVECs as reduced mitochondrial eNOS to regulate the function of blood vessels (Karbach et al. could inhibit HUVEC proliferation and promote The normal ECs express relatively low levels of adhesion apoptosis and necrosis. Soenen et al. 2014. 2015. 2015. Suzuki et al. 2017). therefore. 2014.. whereas pro- cytotoxic effects in HUVECs after TiO2 NP exposure (Suzuki et al. ZnO and TiO2 NPs) (Li et al. 2011. However. viability and membrane integrity. This is also the case in using and tissue factors) and up-regulation of adhesion molecules (e. as peroxynitrite production due to iNOS overexpression were Gong et al.com/journal/jat .. Danielsen et al. Toxicol. 2016). monocyte adhesion to HUVECs is not always correlated with the 2015). 2012. Cao et al. 2015). 2014. It should be noticed. 2015.... 2014. Some studies also showed increased monocyte adhesion to NP-exposed HUVECs. 2014. CB NPs (Vesterdal et al. Cao et al. but it has been suggested the relevant factors (Fig...HUVECs as an in vitro model (Supplementary Information Table 2)..g.. Using this assay. such as silica NPs (Corbalan et al. Guo et al. it should be noticed that particle toxicology studies to detect DNA damage (Moller et al. DNA associated with increased expression of ICAM-1 and VCAM-1 in strand breaks and oxidative modifications of DNA (Frikke. chemokines considered (Azhdarzadeh et al. However.. 2017). and some of the relevant ICAM-1. which can lead to 2011... Klingberg et al. 2011. that the data regarding the cytotoxicity of some types of NPs were inconsistent in literature. Ltd.. 2012. For example.. in a 2014a). Another suggestion is after exposure to a variety of NPs. ZnO NPs.e. 2014.. Guo et al. Guo et al. Appl. which is in agreement with the increased expression of adhesion molecules Genotoxicity (Danielsen et al.. It appears that NPs may affect the function et al. Duan of THP-1 monocytes to HUVECs. MWCNT and micelles based (2015) showed that exposure to Ag. of NPs.. 2011. have also been reported to be cytotoxic to HUVECs. carbon black in vitro observations that NP promoted NO over-production in NPs (Frikke-Schmidt et al... whereas only Ag NPs significantly promoted adhesion Schmidt et al. all of these studies combined indicated that J. interactions. Suzuki et al. Li (Vesterdal et al. Cao et al. Soenen et al. 2) (Libby. The exact reason for the inconsistent reports mediators and express adhesion molecules to recruit monocytes remains unclear. interleukins... Shi et al. 2017. However. CB NPs. 2015. It is.). intracellular Ag accumulation (Shi et al. 2012). Shi et al.. Nevertheless. VCAM-1 and selectins) have been observed in HUVECs factors will be discussed in a later section. 2012. 2016c)... The Comet assay is one of the most popular assays used in 2014. Cao et al. as 2012) and superparamagnetic iron oxide NPs (Astanina et al. Ucciferri et al. Duan et al. inhibited proliferation as well as 2014. Gu et al. eNOS uncoupling which could dissolute to release high levels of Zn ions.. 2015. 2014... an insoluble NP... For example. Endothelial activation For example. Montiel-Davalos et al. Duan et al. (2013a) further showed that silica-NP. HUVECs to assess the toxicity of NPs. In our recent study. wileyonlinelibrary. 2014. have been HUVECs. Similar effects were also observed in HUVECs after exposure to silica NPs (Corbalan et al. 2015).. Gimbrone and Garcia-Cardena. we also found that has been suggested as a protective mechanism to maintain the monocyte adhesion was increased after exposure to ZnO NPs with genome stability in response to DNA damage.. Vesterdal et al. Duan et al. which was supported by the et al. 2013b..) and silica NPs (Corbalan et al. however. 2014a. Xu et al. Montiel-Davalos et al.. observed in HUVECs after exposure to Ag NPs. 2015..... In contrast. the same group showed.. 37: 1359–1369 Copyright © 2017 John Wiley & Sons. TiO2 NPs (Mikkelsen et al.. but the THP-1 monocyte adhesion was checkpoint activation as well as inhibition of proliferation. and monocytes adhere poorly to them. 2011. 2016). metal-based NPs (Ag.. 2013. 2016c.. i. 2012a. HUVECs associated with oxidative stress and inflammation 2014b) and MWCNT (Guo et al. Calarco et al.. which can release the highly cytotoxic Cd 2014). generated from a single assay could be minimized (Soenen et al... so the false-positive or false-negative results 2014b. Blechinger progression in atherosclerotic mice. 1363 toxicity. 2012).... 2017). like other ECs. bioactivity owing to eNOS uncoupling and increased NO as well 2010. (2012) reported that TiO2. Chen et al. later study.. including carbonaceous NPs that it is necessary to use multiple assays to assess NP-cell (CB NPs and carbon nanotubes) (Vesterdal et al. 2016). Danielsen et al. it has been shown that repeated exposure to ions (Yan et al. The authors suggested that it could be because were generally shown to be non-toxic or only slightly toxic of CB NP-induced oxidative stress having altered the signaling (viability of HUVECs larger than 80% after NP exposure) at relatively pathways of the cells or damaged receptors or myofibrils high concentrations (Bartczak et al. which indicated that DNA that NP exposure may affect monocyte adhesion through both damage could be a sensitive marker to reflect NP-induced adhesion molecule-dependent and -independent pathways. possible significant effect on cytotoxicity.. 2013. The associated with the toxicological responses of NPs should be release of inflammatory mediators (e. which not significantly changed. Huo et al..g. 2012. atherogenic stimuli could activate ECs to release inflammatory 2014. such as silica NPs. 2011.. 2011.. 2013a). et al. that CB NPs did not significantly affect NO production although the toxicity of these NPs appears to be more modest in HUVECs but promoted vasomotor dysfunction in artery compared with the soluble. 2016.. sMCP-1 and (2013) showed that micelles based on polyethyleneimine (PEI). 2012. Danielsen et al. could induce DNA damage in HUVECs. Some types of rutile TiO2 NPs could lead to a modest increase in plaque insoluble NPs. Guo et al.. Moreover. 2012). sVCAM-1 was not significantly affected in HUVECs after the PLGA polymer-induced DNA damages in HUVECs without a combined exposure (Gong et al. 2015b). The micelles based on of HUVECs as well as blood vessels both in NO-dependent and - polymers also showed little to no toxicity to HUVECs (Menard independent mechanisms.. Gu et al. the presence of palmitate. 2014. 2015). 2016. 2012. 2013. 2012. Gimbrone and Garcia-Cardena. Cao et al. 2011. we and others did not observe molecules.. Guo et al. Cao et al. (2014b) showed that et al. HUVECs. 2015).. 2016). Danielsen et al. 2011). Montiel-Davalos et al. (Mikkelsen et al..

. dysfunction of organelles could be a possible and membrane (Vesterdal et al. These two studies these studies suggested that NP-induced oxidative stress could indicated a role of ER stress in the toxicity of ZnO NPs to be associated with the adverse effects of HUVECs (Supplementary macrophages but not HUVECs. to NP exposure Supplementary Information Table 2). Yan et al. such as attenuated QD-induced apoptosis. In addition. form a (bio)molecule corona and consequently HUVECs has been observed after exposure to ZnO NPs. 2010. Chen et al.. e. (2014) showed that exposure to ZnO NPs induced the expression Interestingly. which further confirmed the peroxynitrite. as suggested by the three-tier model (Nel to polystyrene NPs was dependent on surface chemistry. 2014a.. which is the key The human blood is rich in proteins and other biomolecules... but the exact role of ER stress activation in NP-induced HUVEC toxicity still needs Atherosclerosis is a chronic inflammatory disease. 2011. 2017)... the of ER cisternae and up-regulation of ER stress markers. to further exacerbate oxidative stress in HUVECs role of ER stress in QD-induced apoptosis. Yan et al. One of the and Ag NPs. NP. 2015). Thus. which can reduce the wileyonlinelibrary. Ltd. Vakifahmetoglu-Norberg et al.. (2009) showed that the affinity of serum proteins inflammatory injury. which desferrioxamine (DFO) could inhibit CB NP-induced intracellular could be further transferred to HUVECs in macrophage– ROS.. following et al. Shi et al. Yu et al. CB NPs. Toxicol. either owing to the activation proteins could competitively absorb into the surface of SWCNT in of NF-κB or oxidative stress. 2011. N-acetylcysteine (NAC) could attenuate the adverse effects of However.. Silica NPs affect the toxicity of NPs to ECs (Docter et al. NPs and serum proteins (Docter et al. it has been shown that the presence of antioxidant of a panel of ER stress markers before inducing apoptosis.. 2011. 2017). 2014)... All of endothelial co-cultures (Chen et al. 2010. Y. thus leading in HUVECs (Liu and Sun. 2012. in our recent study. it has been shown that the an order of COOH > amidine > amine ~ lysine > presence of antioxidants NAC or DFO could also attenuate NP.. Ge et al. Guo et al. At present. Cao et al. 2016... Cao et al. The activation of NF-κB in (bio)molecules.. 2016b. Tsou et al. 2011. The antioxidant systems ( generation of reduced ER is a crucial organelle involved in the regulation of cell survival. Ge et al. which in turn leads to an imbalance between termed as ER stress (Sano and Reed. Cao et al. 2013a. Corbalan et al.. Shi et al. and exposure to further studies. 2017. 2006. In support of the theory. 2016b). activation of macrophages with a ER stress et al. leading to the release of inflammatory mediators and expression of adhesion molecules as discussed earlier (. Rather. 2016.. SWCNT and collapse.. GSH and activities of antioxidant enzymes) could also be inhibited and perturbations of normal function of ER can lead to a condition by NPs in HUVECs.. Shi et al. which could mediate endothelial dysfunction (Liu well-studied examples is the formation of protein corona between and Sun.. 2011. particularly mitochondria and endoplasmic reticulum (ER). regulator of the inflammatory cascade in atherosclerosis (Van der Owing to the high. (2011) showed that the presence of iron chelator inducer promoted the toxicity of ZnO NPs to macrophages. e. 2015). In another study. suggesting a risk of atherosclerosis development in human There are many organelles that are crucial to the function of cells. although exposure to Ag NPs induced cytotoxicity to both types of cells. Moller mechanism associated with the toxicity of NPs to HUVECs..g. Duan et al. 2014. inhibition of ER stress uncoupling. 2015).. stressing HUVECs with ER stress MWCNT. the different affinity to induced inflammatory responses or endothelial activation in proteins was positively correlated with the associations of NPs to HUVECs (Frikke-Schmidt et al. 37: 1359–1369 . dysfunction of organelles. One possible link between The microenvironment NP exposure and inflammatory responses in HUVECs could be the activation of nuclear factor-κB (NF-κB). Mitochondria Mechanisms for the adverse effects are semi-autonomous organelles involved in energy production and perturbed mitochondrial function has been implicated in Oxidative stress CVD (Silva et al. 2010. 2014. In another study. Another possibility is that NP-induced oxidative stress promoted Ehrenberg et al.. DNA. Appl. Huo et al. 2015). (2011) also showed that different serum induced inflammatory responses. Moller et al. beings after long-term exposure (Supplementary Information In recent years. promoted mitochondrial membrane potential (MMP) 2013. 2011. Yan inducer did not significantly affect the toxicity of ZnO NPs (Gu et al. as the molecular mechanisms of the toxicity to ECs (Supplementary Information Table 3).. CdTe QD. 2013). NPs has been suggested to promote atherosclerosis development by inducing inflammation (Cao et al. Some of the NPs. exposure of HUVECs to NPs could lead to endothelial activation Dysfunction of organelles in vitro. Ag NPs and QD to HUVECs (Guo et al. they could easily absorb Heiden et al.com/journal/jat Copyright © 2017 John Wiley & Sons. Meanwhile. Guo et al. transferrin > bovine serum albumin. showed that Ag NPs induced ER stress responses in lung cells but not HUVECs. 2013). cysteine ~ methyl > PEG.. 2015).g. for example silica NPs and mechanisms responsible for NP-induced toxicity (Donaldson et al. Moreover. 2014). it has been shown that NPs could activate Factors to influence the responses of HUVECs HUVECs in vitro. It Oxidative stress has been considered as one of the most important has been shown that exposure to NPs. to the oxidative damage of biological molecules. showed that CdTe QD induced ER stress in HUVECs as dilatation 2015. 2014). along with oxidative milieu imposed by NP exposure can also result in eNOS the activation of apoptosis. Interestingly. (2015) Information Table 3). 2017). 2015. (2016) oxidant generation and antioxidant capacity (Guo et al. J. could be associated with NP-induced the order of bovine fibrinogen > gamma globulin- 1364 adverse effects in HUVECs. have been shown to be chemically active that could release of mitochondrial cytochrome c and cleavage of caspase directly induce ROS in acellular conditions or HUVECs. et al. free energy of NPs. some studies indicated that NPs might induce the Table 2). 2016). (Duan et al. 2016c). 2010. Similarly. Thus. which in turn generate the oxidants. Similarly.. 2013b. DNA damage and expression of adhesion molecules... Frikke-Schmidt et al. it seems that some Inflammation types of NPs could induce ER stress in HUVECs. Cao et al. HUVECs.... proteins 2013a). Duan et al. which consequently induced apoptosis owing to the MWCNT.

whereas the uptake of anti- showed that the formation of protein coronas on the surface of ICAM-1-coated Au NPs was increased in HUVECs exposed to flow Au and Ag NPs (citrate and lipoic acid coated) by human serum and TNFα. wileyonlinelibrary. For (Hotamisligil and Erbay. 2008.... 37: 1359–1369 Copyright © 2017 John Wiley & Sons.HUVECs as an in vitro model cytotoxicity of SWCNT to HUVECs. Similar effects were observed in HUVECs exposed to of NPs to HUVECs.1 and the most effective. 2016). For example. could under inflammatory conditions when assessing the toxicity of absorb NPs. In a later study.. but not by fibrinogen. (2010) showed oxidative stress (Cao et al.. atherosclerosis is a typical inflammatory NPs could influence the biological responses of NPs to HUVECs (. The anatase form of Under in vivo conditions. In contrast. significantly increased the interactions of NPs with HUVECs and (2015a) showed lower internalization of unmodified Au NPs in reduced the toxicity.. 2016). Ltd. to that metal-based NP-induced cardiovascular effects were better predict the response of ECs to NPs in vivo. Samuel et al.. but and Yamamoto. Gong et al. Under stretch. Appl. Ge et al. Similarly. we showed that ZnO NPs enhanced TNFα-induced ICAM-1 expression in that the presence of palmitate could coat ZnO NPs but did not HUVECs. Tenzer 0.. Sasidharan et al. intracellular ROS. 2017).. the time-dependent protein corona formation with that measured under static conditions. size and crystal structure but not factors that can make the diversity of ECs in a surface coating or charge (Danielsen et al. recent study showed the accumulation of intracellular Zn ions a saturated fatty acid.com/journal/jat . there inflammatory responses or endothelial activation in HUVECs was a relatively higher cytotoxicity in HUVECs after the combined (Wischke et al. We have NPs. microenvironment can lead to the diversity of ECs. (2011) showed shear stress-dependent cytotoxicity of silica expression in HUVECs.. (2015) HUVECs after adaptation to flow.. 2011).. One of the examples is shear stress or short and long MWCNT induced cytotoxicity.. 2016c). This is also health effects (such as inflammation) in metabolic diseases the case for HUVECs (Supplementary Information Table 4). However. (2015) compared the effects of a panel of metal-based NPs to HUVECs. A pilot study by Kim the longer one was associated with higher adhesion molecule et al. 2015b). The coating of ZnO NPs or charge of TiO2 NPs showed The diversity of ECs under in vivo conditions no effect to influence their toxicity to HUVECs. with bovine fibrinogen being of 0. Both (Setyawati et al. (2015) showed that the toxicity of Au NPs to HUVECs and polystyrene NPs after incubation with human plasma. 2013. Danielsen et al. All of these studies albumin or immunoglobulin corona showed a higher association indicated that shear stress or stretch could modulate the uptake with HUVECs (more binding to the membrane or internalization) and toxicity of NPs to HUVECs. Gimbrone and Garcia-Cardena. As a consequence. Gregor and Hotamisligil. 2016a). 2015). the physiological and/or pathological TiO2 NPs showed a stronger effect compared with the rutile one. 2015). In the future. These two studies indicated a role of structures and shapes J. All of these studies The diversity of ECs under diseased conditions should also be in combination indicated that the formation of protein corona on considered. NPs with human serum cytotoxicity or inflammatory responses. it has been shown that the presence of palmitic acid. 2016). it is necessary to consider the diversity of ECs recently proposed that excessive nutrients. our For example. 2015). The assessment of the combined effects of excessive nutrients and NPs to HUVECs The physicochemical properties of NPs may better predict the toxicity of NPs to ECs in vivo especially in the context of metabolic diseases because it has been suggested It is well accepted that the physicochemical properties of NPs can that the presence of excessive nutrients can mediate the adverse define their interactions with cells (Beddoes et al. Similarly. In a recent study. this issue may showed that the uptake of poly[acrylonitrile-co-(N- be of more relevance in assessing the toxicity of NPs to HUVECs vinylpyrrolidone)] [P(AN-co-NVP)] NPs into HUVECs was enhanced than using any other cell lines. that the inflammatory stimuli might affect the uptake and toxicity 2016). Klingberg et al. coated MWCNT and promoted MWCNT. which was associated with particle endocytosis owing to et al. 2015. 300 different proteins was rapidly formed (< 0. on the uptake of Au NPs (Klingberg et al. by inflammation induced by the treatment of IL-1β. disease (Libby. Cao et al. Toxicol. 2016). Ucciferri et al. Chistiakov et al. induced by ZnO NP exposure was not significantly affected by induced monocyte adhesion to HUVECs without an effect on the presence of LPS (Gong et al. compared with those with fibrinogen corona. 1365 showed a higher uptake of CdTe QD and silica NPs at a shear stress 2014b). was approximately 20% lower under flow conditions compared Furthermore.. and Supplementary Information Table 4). Besides proteins.5 min) on silica Fede et al. like proteins. (2013) revealed that a complex protein corona with almost the increased formation of actin-based cytoskeletal structures. stabilized uptake of silica NPs into HUVECs without influence on NP-induced NPs in suspension. 2017). example. 2016a). The authors suggested (Eelen et al. Gimbrone and Garcia-Cardena. (2014) showed a reduced albumin or immunoglobulin. change the physicochemical properties of NPs and NPs to HUVECs. depending on the way of stimuli and types of MPEG-PLA-based micelles and palmitate (Liu et al. (2012) smaller compared with those induced by MWCNTs (Cao et al.05 Pa compared with the uptake at a shear stress of 0. These studies indicated exposure owing to the intrinsic toxicity of palmitate (Gong et al.5 Pa. (2013) However. stretch. Freese et al. which can influence the functional properties of ECs (Ando expression of adhesion molecules and monocyte adhesion. whereas we and others did not find synergistic effects significantly affect ZnO NP-induced monocyte adhesion or the of the presence of inflammatory stimuli (IL-1β or LPS) and NPs on release of inflammatory cytokines in HUVECs. NPs (Supplementary Information Table 4). Thus. some of the dependent on NP composition. 2015.. similar to what have been the inflammatory stimuli may affect the responses of HUVECs to observed in other cell lines (Docter et al. Wischke et al. (2014) also showed that that exposure of carbon black (CB) NPs could induce similar effects HUVECs were more susceptible to Ag NP-induced cytotoxicity to HUVECs. the interactions between NPs and excessive activation of HUVECs by TNFα or LPS showed a slight or no effect nutrients may also need further investigations (Cao et al. In our recent study. Using label-free snapshot proteomics. the same group showed NPs to HUVECs. 2011. making them There was also a stronger response of HUVECs to larger TiO2 NP completely different from the conventionally cultured ECs in vitro exposure compared with the smaller one. as ECs directly contact serum proteins.. 2012. 2017. but the responses except ROS generation appear to be and inflammation under flow conditions. 2015).. There were several findings from that study. physiological/pathological conditions should be considered (2014a) compared the effects of different length of MWCNT. Tsou et al. eventually affect the toxicity (Cao et al.

The results of these studies indicated that NPs could was not significantly affected after exposure to ZnO NPs or TiO2 be internalized into HUVECs by endocytosis pathway as well as NPs (Gong et al. Y. J.. HUVECs to ZnO NPs but not TiO2 NPs promoted the release of extensive studies have used HUVECs as a model to investigate sMCP-1. 2015) physiological stretch conditions could change the responses of ECs to NPs Inflammatory stimuli Different NP uptake The diversity of ECs under pathological (Setyawati et al. Modeling the Blood- Conclusions Brain Barrier in a 3D triple co-culture microfluidic system. 2016. These factors and their implications in future nanotoxicology studies are summarized in Table 1 (the The authors did not report any conflict of interest. Cao et al. 2013. Soc. ECs covering blood vessels are of particular Ando J. Acad. However. detailed each research example is summarized in Supplementary Information Table 4).. all the three studies showed that the soluble genotoxicity. Kamm RD. wileyonlinelibrary. Consideration of these factors may better predict the toxicity of Conflicts of Interest NPs to endothelium in vivo. used. cytotoxicity and oxidative stress in HUVECs conditions better when using this model.. 2014. Nevertheless. Pavesi A. 2016a) Changed NP uptake and could affect the behavior of NPs in toxicity circulation Shear stress or Different NP uptake and toxicity The diversity of ECs under physiological (Setyawati et al.. by comparing the responses of ECs to a series of NPs. 2017). inflammatory HUVECs associated with increased intracellular Zn ions. Ltd. The rapid development of nanotechnologies can lead to Agarwal R. Suzuki et al. 2017). and it is cells preferentially internalize hydrogel nanodiscs over nanorods and use shape-specific uptake mechanisms. a number of factors could influence EC–NP start-up grant (15QDZ47). 37: 1359–1369 . (2014) showed that exposure of the regulation of blood vessel function. necessary to understand the effects of NPs to relevant cells lining 110: 17247–17252. In combination. 2014b). the EC–NP interactions. Gu et al. (2013) showed higher ECs to NPs to understand the effects of NPs under in vivo cellular interactions. 2015: 338–341. the toxic effects of NPs on ECs and the in our recent study. induced the cytotoxicity of could be explained by NP-induced oxidative stress. Yamamoto K. Excessive nutrients in chronic diseases (Cao et al. Proc. these studies suggested that the this model to further predict and understand the biological physicochemical properties. 2015) and inflammation conditions could change the responses of ECs to NPs Physicochemical Different NP uptake and toxicity NPs with different properties could show (Azhdarzadeh et al. some studies indicated a role of solubility of NPs (Suzuki et al... Proc. e.. Gong have also evaluated the factors that can influence the responses of et al. expression of ICAM-1 and monocyte adhesion. human blood vessels. shape. IEEE Eng. Roy K. but not insoluble TiO2 NPs. 2015. the release of sMCP-1 or monocyte adhesion mechanisms. Xiangtan University grant (15XZX19) and Xiangtan University In summary. 2016. charge could define the responses of HUVECs (and probably also other types of ECs) to NP exposure. solubility and surface responses of ECs to NPs in the future. Physicochemical properties of NPs should be determined along with the toxicological studies of carbon-based NPs in influencing the effects to HUVECs (Cao ECs serve as the first contact with NPs and play a pivotal role in et al. Shi L. In the future. 2011.. Huhn et al. Appl. eNOS uncoupling and endothelial activation. and it is expected that more studies will use charge. Jurney P.g. Singh V. A summary about the factors that can influence NP–EC interactions and the implications in using HUVECs as a model to assess the toxicity of NPs to ECs Factor Influence Implications References Serum proteins Formation of protein corona. Exposure of HUVECs to NPs could induce cytotoxicity. S.. Gu et al. Ma D. 2015) Changed NP uptake and different from the naked NPs toxicity Excessive nutrients Coating effects on NPs. References Adriani G. 2017. which ZnO NPs. 2015) properties of NPs different toxicity to ECs. Goh EL. Toxicol. A. Med. size. Antioxid. Redox Signal 15: 1389–1403. Conf. The different data obtained transported across HUVECs by exocytosis and paracellular by different groups could be due to the different properties of NPs pathways. The behavior of NPs in vivo could be (Docter et al. Sci. Mammalian increasing contact with human blood vessels with NPs. 2014a. For these reasons. Natl. interactions and should be considered when using HUVECs as the in vitro model to assess the toxicity of NPs to ECs. These studies suggested after exposure to the positively charged Au NPs compared with that HUVECs could be considered as a relatively reliable and simple the negatively charged ones.. which response and dysfunction of organelles.com/journal/jat Copyright © 2017 John Wiley & Sons. In addition. Sreenivasan SV. Biol. it is still necessary Acknowledgements to further understand the exact role of the physicochemical properties of NPs in influencing the biological responses of ECs This work was financially supported by The Scientific Research Fund of Hunan Provincial Education Department (16C1551). which suggested a role of surface in vitro model for ECs.. U. Effects of shear stress and stretch on 1366 interest to nanotoxicological and nanomedicine studies because endothelial function. Table 1.

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