Titanium dioxide nanomaterials cause endothelial cell leakiness by disrupting the homophilic interaction of VE-cadherin

Abstract

The use of nanomaterials has raised safety concerns, as their small size facilitates accumulation in and interaction with biological tissues. Here we show that exposure of endothelial cells to TiO₂ nanomaterials causes endothelial cell leakiness. This effect is caused by the physical interaction between TiO₂ nanomaterials and endothelial cells' adherens junction protein VE-cadherin. As a result, VE-cadherin is phosphorylated at intracellular residues (Y658 and Y731), and the interaction between VE-cadherin and p120 as well as β-catenin is lost. The resulting signalling cascade promotes actin remodelling, as well as internalization and degradation of VE-cadherin. We show that injections of TiO₂ nanomaterials cause leakiness of subcutaneous blood vessels in mice and, in a melanoma-lung metastasis mouse model, increase the number of pulmonary metastases. Our findings uncover a novel non-receptor-mediated mechanism by which nanomaterials trigger intracellular signalling cascades via specific interaction with VE-cadherin, resulting in nanomaterial-induced endothelial cell leakiness.

ARTICLE

Received 28 Sep 2012 | Accepted 27 Feb 2013 | Published 9 Apr 2013

M.I. Setyawati1, C.Y. Tay1, S.L. Chia1, S.L. Goh1, W. Fang1, M.J. Neo1, H.C. Chong2, S.M. Tan2, S.C.J. Loo3, K.W. Ng3, J.P. Xie1, C.N. Ong4,5, N.S. Tan2,6 & D.T. Leong1

The use of nanomaterials has raised safety concerns, as their small size facilitates accumulation in and interaction with biological tissues. Here we show that exposure of endothelial cells to TiO2 nanomaterials causes endothelial cell leakiness. This effect is caused by the physical interaction between TiO2 nanomaterials and endothelial cells adherens junction protein VE-cadherin. As a result, VE-cadherin is phosphorylated at intracellular residues (Y658 and Y731), and the interaction between VE-cadherin and p120 as well as

b-catenin is lost. The resulting signalling cascade promotes actin remodelling, as well as internalization and degradation of VE-cadherin. We show that injections of TiO2 nanomaterials cause leakiness of subcutaneous blood vessels in mice and, in a melanoma-lung metastasis mouse model, increase the number of pulmonary metastases. Our ndings uncover a novel non-receptor-mediated mechanism by which nanomaterials trigger intracellular signalling cascades via specic interaction with VE-cadherin, resulting in nanomaterial-induced endothelial cell leakiness.

1 Department of Chemical and Biomolecular Engineering, National University of Singapore, Singapore 117576, Singapore. 2 School of Biological Sciences, Nanyang Technological University, Singapore 637551, Singapore. 3 School of Materials Science and Engineering, Nanyang Technological University, Singapore 639798, Singapore. 4 Saw Swee Hock School of Public Health, National University of Singapore, Singapore 117597, Singapore. 5 NUS Environmental Research Institute, National University of Singapore, Singapore 117411, Singapore. 6 Institute of Cell and Molecular Biology, Agency for Science, Technology and Research A*STAR, Singapore 138673, Singapore. Correspondence and requests for materials should be addressed to D.T.L. (email: mailto:[email protected]

Web End [email protected] ).

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DOI: 10.1038/ncomms2655

Titanium dioxide nanomaterials cause endothelial cell leakiness by disrupting the homophilic interaction of VEcadherin

ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms2655

The pervasive use of nanomaterials (NM) generated public concerns about their possible health effects and has prompted much scientic investigation1. The new science

of nanobiology requires greater understanding in the interaction of nanoscale particles with biological components. Due to their nano dimensions, it is possible that some of these NM enter into the circulatory system and accumulate in major organs2. Endothelial cells line blood vessels in many highly vascularized organs and function as gatekeepers for nutrients, waste and cell movement. The subtle control of blood vessel integrity is so important that its dysfunction is implicated in tumour angiogenesis and metastatic migration3,4. Much of this control occurs right at the adherens junctions between endothelial cells. These adherens junctions do not merely juxtapose neighbouring cells but act as intracellular signalling conduits determining cell position, proliferation, apoptosis and vascular homeostasis5. When this junctional continuity is disrupted, the space between adjacent endothelial cells increases to at least 12 mm, resulting in endothelial cell leakiness (ECL) or in an in vivo setting, vascular leakiness3. Species like vascular endothelial growth factor (VEGF), thrombin, angiopoietin-like 4 protein4 and histamine5 can perturb this tightly regulated intercellular and intracellular homeostasis and result in ECL. Toxic metal oxide NM are known to be endocytosed and induced oxidative stress6,7; indirectly leading to ECL8. However, we postulate a novel mechanism where ECL can occur without uptake of NM but by virtue of their small dimensions, NM can migrate into and disrupt the adherens junction between endothelial cells. This postulated mechanism would also suggest a novel non-receptor-binding mechanism that directly causes ECL.

ResultsTiO2NM induce ECL. We observed that a relatively benign and commonly found TiO2NM actually caused ECL of an otherwise conuent layer of endothelial cells. We tested commercially available spherical-shaped TiO2NM with primary particle size of 23.5 nm (Supplementary Fig. S1). Hydrodynamic size measurement of TiO2NM showed that these NM formed small aggregates with an average size of 57.1 nm in complex cell culture medium (Supplementary Fig. S1). NM disaggregate and shed off materials when in complex aqueous environments9. This etches the aggregates down to smaller sizes10. Though generally considered to be of low cytotoxic risk6, we observed that TiO2NM with concentration as low as 10 mM induced ECL (red arrowheads) within a short 30 min of exposure (Fig. 1a). This NM-induced ECL effect was sustained after 60 min of treatment (Fig. 1a). On the contrary, micro-sized TiO2 (680 nm) at the same dosage range and exposure duration did not cause any observable disruption of endothelial cell layer (Supplementary Fig. S2), thus supporting the notion that the observable ECL is highly dependent on primary particle size. To quantify leakiness, we plated a conuent layer of endothelial cells over 0.4 mm pores

Transwell inserts and treated the cells with either TiO2NM(23.5 nm) at various concentrations, TiO2 microparticles (680 nm) and 2.5 mM EDTA (as positive control for endothelial cells leakiness). We again observed signicant levels of leakiness when treating the cells with TiO2NM as low as 10 mM concentrations (Fig. 1b) but not in the TiO2 microparticles group (Fig. 1b). This size effect difference is also reported by comparative cytotoxicity studies between ne and ultrane materials11 but not as the cause of ECL. We tested two other similarly sized NM, silicon dioxide and silver, and observed that these two other NM also caused ECL (Fig. 1c). As ECL occurs at the adherens junction, one would logically deduce that the entities holding the adherens junction might be critically affected either

directly or indirectly by TiO2NM. Each neighbouring endothelial cell contributes one arm of VEcadherin dimer to form a homophilic trans-interacted VEcadherin complex. A series of these VEcadherin complexes aligns itself to form a pericellular zipper-like structure along an intact inter-endothelial cell junction border5. Where ECL has occurred coincided with the loss of this zipper-like structure (Fig. 1a). The observation that TiO2NM caused microscopic ECL further reinforced the notion that the homophilic interacted pairs of VE-cadherin were disrupted. This size factor of NM provided some clue to the mechanism. Uncovering the mechanism of how NM induce ECL would have signicant biological signicance as ECL is involved in cancer metastasis and progression3, edema and diabetic retinopathy12.

Induced ECL is independent of oxidative stress or apoptosis. The canonical perpetrator of NM-induced toxicity is oxidative stress6. Metal oxide NM, like ZnO and CuO, can induce elevation of intracellular reactive oxygen species (ROS)6, which indirectly cause ECL through cytoskeletal damage8 or indirectly by activating apoptotic events, resulting in cellular shrinkage. Therefore, initially we checked whether TiO2NM-induced ROS generation may have caused ECL. However, we observed signicant elevation of ROS only after a time period (Fig. 2a) that far exceeds our initial observation of ECL at a very brief TiO2NM treatment time of 30 min (Fig. 1a).

A hallmark manifestation of apoptosis is cellular shrinkage from neighbouring cells13. Early report showed that TiO2NM could trigger the apoptotic pathway in another cell type, mouse epidermal JB6 cells, after 180 min of TiO2NM treatment14;

which is far later than our initial detection of ECL at 30 min. So we again checked whether apoptosis triggered by TiO2NM caused ECL. Based on non-cleavage of important caspases (Fig. 2b), like caspases 3, 8 and 9, and also poly (ADP-ribose) polymerase, we concluded that apoptosis cannot be a cause for this type of ECL.

As intracellular processes, such as ROS generation and apoptosis, cannot explain the observed ECL, we postulate that the trigger might be pre-endocytotic or extracellular. Using TiO2

NM covalently tagged with uorescein isothiocyanate (FITC) and monitoring its time-dependent endocytosis, we observed signicant uptake of TiO2NM 180 min after exposure (Fig. 2c), noticeably far later than our ECL observation at 30 min post NM treatment (Fig. 1ac). Next, we quantied the cellular uptake of TiO2-FITC in membrane and cytosolic fractions, and showed that

TiO2NM was mostly in the membrane fraction within the time scale of ECL (Fig. 2d). In order to further show that uptake is not necessary for the occurrence of the ECL, we blocked endocytosis with 10 mM monodansylcadaverine (MDC) or 5 mM methyl-bcyclodextrin (MbCD), and that did not prevent ECL induced by 50 mM TiO2NM (Fig. 2e). The same phenomenon was observed when cells were treated at a higher concentration of 250 mM TiO2NM instead (Supplementary Fig. S3). Control experiments for endocytosis inhibition (Fig. 2e) showed that endocytosis was indeed inhibited and at 30 min there was also insignicant uptake of TiO2-FITC (Fig. 2e). Collectively, our data strongly suggest that the cause of this ECL must have occurred before any signicant TiO2NM endocytosis and that the observed ECL is of extracellular and membraneous origin.

TiO2NM directly bind VEcadherin in adherens junctions. We observed that early ECL is not dependent on endocytosis (Fig. 2ce) and TiO2NM treatment profoundly resulted in leakiness while TiO2 microparticles did not (Fig. 1a,b). Considering the known biology of adherens junctions, we reasoned from our experimental evidences and hypothesized that TiO2NM by

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Figure 1 | TiO2NM induce ECL in vitro. (a) TiO2NM induced ECL within 30 min of exposure and this effect persisted for at least 60 min of exposure. ECL (red arrowhead) was observed on a conuent monolayer endothelial cells treated with various concentrations of TiO2NM. Higher magnication windows showed leakiness between cells. Adherens junctions VEcadherin visualized with immunouorescence (green) and nuclei by 40,6-diamidino-2-phenylindole (blue). Scale bar, 50 mm. (b,c) ECL semiquantitative transwell assay showed increased leakiness after treating with

TiO2NM (23.5 nm), SiO2NM (15 nm) and AgNM (20 nm). Data are meanss.d., n 3. Students t-test, *Po0.05.

virtue of its size may have migrated into the nano-range size of the adherens junction (determined minimally by the length of a homophilically interacted complex of cadherin of B22.5 nm.15 It then directly binds to and disrupt the extracellular homophilic interaction of VEcadherin, the key molecule that maintains endothelial cellcell integrity.

We tested our direct binding hypothesis and the necessity of an intact adherens junction (a priori condition for any intact cell-cell contact) by modifying a standard protein pull-down assay but using TiO2NM as a precipitation agent (Fig. 3a). To determine whether intact adherens junctions are necessary and to control for the artefactual situation where binding of VEcadherin with TiO2NM has instead occurred in lysis condition, we included a group where protein extracts were only exposed to TiO2NM after cells were lysed (P, post-lysis group). VEcadherin was indeed pulled down by TiO2NM in a dose-dependent fashion, while the other controls (no-beads control (C) and agarose mock pulled-down control (A), post-lysis control (P) and TiO2 microparticles-treated group) did not (Fig. 3a). Tight junction

protein claudin-5, found in inter-endothelial niche, however, was not pulled down by TiO2NM (Fig. 3a). Intracellular proteins, like SOD1 and tubulin, were not pulled down as well (Fig. 3a). This suggests some specicity of TiO2NM towards VEcadherin and again suggests involvement of the adherens junction. The observations that the post-lysis TiO2 treatment group (P)

and microparticle-treated group did not pull down any detectable VE-cadherin suggest that the in situ juxtaposition of VEcadherin within an intact adherens junction is required for this binding and it is not merely direct physical interaction between VEcadherin and TiO2NM in a post-lysis scenario. We further showed that there is a physical interaction in situ between VEcadherin and TiO2NM by modifying a relatively new molecular interaction technique called proximity ligation assay (PLA), used mainly to prove direct proteinprotein interaction (Supplementary Fig. S4). We showed with modied PLA protocol that IgG-conjugated TiO2NM interacted with VEcadherin (o40 nm apart) physically (Fig. 3b). Implicitly observed in the images (Fig. 3b) is that this interaction is along the

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Figure 2 | TiO2NM-induced ECL is independent of ROS formation, apoptosis and endocytosis (a) Signicant levels of ROS upregulation in the TiO2 NM-treated cells were detected far later than the 30 min when leakiness was observed. H2O2 (200 mM, 2 h) served as positive control. Graded shaded triangles represent increasing time points of 0, 0.5, 1, 2, 4, 8, 12 and 24 h. Data are meanss.d. n 3. Students t-test compared with untreated control,

*Po0.05. (b) Immunoblotting analysis of apoptosis markers showed no evidence of apoptosis over various concentrations of TiO2NM and time points. (c) FITC-conjugated TiO2NM (1,250 mM) were used to visualize the uptake of TiO2NM into endothelial cells. Merged phase contrast and confocal images showed there was signicant uptake (180 min) only much later than the time of ECL (30 min, Fig. 1a). Scale bar, 25 mm. (d) Cells were treated with 250 mM

TiO2FITC and the membrane and cytosolic fractions TiO2FITC content measured. Normalized to cytosolic fraction at t 0. There were consistently

increasing amounts of TiO2FITC associated with the membrane fraction up to 60 min. Data are meanss.d. n 3. Students t-test, compared with

corresponding cytosolic sample #Po0.05; compared to t 0 group, *Po0.05. (e) Blocking endocytosis with inhibitors did not prevent leakiness.

Red arrowheads indicate the presence of ECL on endothelial cells monolayer. Scale bar, 50 mm. Data are meanss.d. of n 3. Students t-test, compared

with no-inhibitor control, *Po0.05.

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adherens junction region and occurred in situ and not in the lysed state. To further prove the distance sensitivity of the PLA technique, the negative control pair, VEcadherin and actin which are close by but separated by a-catenin5 complexes were utilized, and as predicted no positive PLA signals was observed (Fig. 3b).

TiO2NM trigger VEcadherin pathway and actin remodelling. TiO2NM binding (Fig. 3a, b) only partially explains the ECL phenomenon. Disruption alone does not account for gaps that were at least of micron separation distances as seen in the confocal images of leakiness (Fig. 1a and Fig. 3b). Activation of the VEcadherin-mediated actin-rearrangement pathway explains the size of observed gaps. Based on the canonical VEcadherin disruption pathway, an external ligand stimulus like VEGF or histamine activates phosphorylation of intracellular domain of VEcadherin at its Y658 and Y731 residues16,17. Perimembraneous actin stability is linked to VEcadherins intracellular domain through interactions with b-catenin and p1205 via these two phosphorylation sites.

Cadherincateninsactin ternary complex has a pivotal role in

cell shape and junction stability and endothelial permeability5. The phosphorylation at Y658 and Y731 signals the loss of VEcadherin interaction with p120 and b-catenin, respectively, consequentially activating the actin remodelling pathway followed by the deformation of cell shape resulting in gaps between cells5. The manner which NM induced endothelial leakiness (NanoEL) appeared to be very different because it is unlikely that endothelial cells have evolved a cognate receptor for an articial entity like a nanoparticle to bring about the NanoEL effect. Therefore, we asked to what degree of similarity does NM-induced endothelial leakiness coincide with the canonical ligandreceptor axis. We then showed that TiO2NM treatment also resulted in phosphorylation outcomes reminiscent to that of VEGF and histamine (Fig. 4a). Guided by our VEGF- and histamine-mediated signalling data, we checked with a Src kinase inhibitor, PP1, whether Src kinase might be responsible for this TiO2-NM-induced phosphorylation (Fig. 4a). The inhibition of

Src kinase with PP1 did not prevent the phosphorylation of the Y658 residue of VEcadherin while the phosphorylation of the Y731 residue instead was repressed. As expected, inhibiting Src kinase with PP1 reduced the endothelial leakiness induced by VEGF and histamine16,17 but did not have a signicant effect on

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Figure 3 | TiO2NM directly bind to adherens junctional homophilic VEcadherin. (a) The presence of TiO2NM pulled down VEcadherin (VEC) in a dose-dependent manner. Post lysis (P), addition of TiO2NM (1,250 mM) to a non-treated control did not pull down any detectable VEcadherin.

This suggested that an intact adherens junction is important for TiO2NM to bind to VEcadherin. This showed that TiO2NM binds to VEcadherin within the adherens junction and not in the lysis buffer condition. Protein A Agarose beads (A) were added to show that no detectable VEcadherin were precipitated without addition of TiO2NM. Whole cell lysate showed similar expression of VEcadherin across the various groups, which indicated that

TiO2NM did not affect cellular VEcadherin expression transcriptionally or post-translationally. Schematic cartoon showing the experimental setup.(b) Direct interaction between TiO2NM and VEcadherin was assessed with modied PLA (Supplementary Fig. S4). Intact monolayer of endothelial cells was treated with mouse-IgG-conjugated TiO2NM for 30 min. Through the PLA protocol, red uorescence signals were observed following mouse

IgG-conjugated TiO2NM treatments (100 and 250 mM), thus showing direct interaction. Untreated control, NM nonspecic control and non-interacting proteins (1 antibodies: VEcadherin actin; V A) controls all showed negative signals. Quantication of red signals showed increased TiO2NM

interaction with VEcadherin with increasing dose of TiO2NM. Scale bar, 50 mm. Data are meanss.d. of n 3. Students t-test, compared with untreated

control, *Po0.05.

TiO2NM-mediated leakiness (Fig. 4a). These observations suggested that a non-Src tyrosine kinase might be involved. Subsequently, immunoprecipitation results showed the loss of interaction between VEcadherin with either b-catenin or p120 catenin as early as 15 min of TiO2NM treatment (Fig. 4b). We next checked for internalization and degradation of VEcadherin; events that are linked to the leakiness phenotype18. Consistent with observations from the treatment of VEGF and histamine, TiO2NM stimulated the internalization of VEcadherin (Fig. 4c)

and its subsequent degradation (Fig. 4c). As observed earlier with the modied PLA technique, there were TiO2NM interacting with VEcadherin further inward from the cell surface (Fig. 3b). This suggested that TiO2NM after binding with VEcadherin might have followed VEcadherin into the cytoplasm during the process of internalization of VEcadherin. Taken together, we proposed that the disruption of homophillic interaction of VEcadherin by TiO2NM would trigger the machinery that leads to actin rearrangement resulting in changes to cell shape and therefore leakiness between neighbouring endothelial cells. To investigate whether perturbation of the cytoskeletal network can prevent TiO2NM-induced leakiness, conuent layer of endothelial cells were pretreated with the RhoA kinase (ROCK) inhibitor, Y-27632 (5 and 10 mM), for 1 h. ROCK inhibits depolymerization of F-actin by myosin phosphotase and colin,

resulting in the F-actin stabilization19. The inhibition of ROCK activity reduced the formation of stress bres while maintaining the cellcell adhesion, as shown by the VEcadherin staining, without any apparent observable formation of intercellular gaps (Fig. 4d). In fact, transwell assay suggested that Y-27632 treatment actually enhanced endothelial barrier function by about twofold presumably via recruitment of F-actin to a- or bcatenin-containing junctions20. Interestingly, TiO2NM treatment to Y-27632 preconditioned samples still led to an increase in dextranFITC ux, albeit to a much lower level compared with the untreated Y-27632 control (Fig. 4d). These data showed that the perturbation of the cytoskeletal network was able to mitigate but unable to completely block TiO2NM-induced ECL. This suggests that cytoskeletal remodelling is necessary and potentially a downstream event that facilitates the formation of TiO2NM-induced micron-sized gaps. Overall, it was unexpected that TiO2NM binding to VEcadherin itself can trigger phosphorylation of VEcadherin because a ligand receptor mechanism was previously thought to be the trigger for the same phosphorylation outcomes. Our data further showed that the binding of a foreign entity, like a nanoparticle, can trigger the same outcome as VEGF or histamine, albeit through a yet unclear kinase-initiated cascade. This can still culminate to ECL.

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Figure 4 | TiO2NM induce phosphorylation, internalization and degradation of VEcadherin. (a) TiO2NM treatment induced tyrosine phosphorylation of VEcadherin at Y658 and Y731 but may not involve Src kinase. TiO2NM (250 mM) triggered important Y658 and Y731 phosphorylation. VEGF (V), histamine (H) and EDTA (E) served as control stimulants. Src kinase inhibitor, PP1, effectively inhibited VEGF- and histamine-induced phosphorylation of

VEcadherin at Y658 and Y731. However, TiO2NM treatment showed persistent phosphorylation of Y658 even with PP1 treatment. PP1 treatments were able to reduce the degree of FITCdextran ux across the endothelial monolayer in VEGF- and histamine-treated samples. However in TiO2NM case, dextran ux was relatively less repressed with PP1 treatment as compared with PP1 treatment in VEGF and histamine groups. Data are meanss.d. (n 3).

Student t-test, Po0.05 (*treated versus untreated control; #with PP1 versus without PP1). (b) TiO2NM treatment induces release of p120 and b-catenin from VEcadherin. Decreased interaction between VEcadherin with b-catenin and between VEcadherin with p120 following TiO2NM treatment (250 mM). The vice versa immunoprecipitation of b-catenin also show decreased interaction with VEcadherin. (c) TiO2NM mediated internalization and degradation of VEcadherin. Increased amount of internalized VE-cadherin was observed following TiO2NM treatment. Potassium depletion (K depl.) and MDC further reduced internalization of VEcadherin. Immunouorescence helps to detect internalized VEcadherin (white arrowheads) following treatment of TiO2NM, VEGF or histamine, while for control group, VEcadherin was conned to the cell boundaries. The cell periphery is demarcated by the dashed line. Scale bar, 15 mm. TiO2NM treatment led to the lysosomal degradation of VEcadherin. Inhibiting the proteasome pathway with MG132 did not result in any signicant increase in the amount of VEcadherin. In contrast, inhibiting the lysosomal pathway with chloroquine (CHQ) showed an increased stability of internalized VEcadherin as shown in the western blot data and the confocal imaging of internalized VEcadherin. (d) TiO2NM activated actin remodelling that led to leakiness. ROCK inhibitor, Y-27632, treatment alone reduced inherent endogenous endothelial leakiness and negated

TiO2-NM (250 mM)-induced leakiness. Treatment with Y-27632 for 1 h effectively abrogated formation of stress bres without comprising endothelial barrier integrity as shown by the VEcadherin staining. Scale bar, 20 mm. Data are meanss.d. (n 3). Students t-test, Po0.05. #Signicant difference

against TiO2NM treatment.

TiO2NM cause ECL in subcutaneous blood vessels. In vivo ECL can lead to edema owing to an increase in interstitial pressure. In extreme cases of ECL, cellcell junctions are chronically disrupted resulting in extensive vascular damage. We chose two distinctly different mouse models to investigate whether TiO2NM causes leakiness in an in vivo setting. To show that there is direct vascular leakiness, we injected either TiO2NM or vehicle buffer into subcutaneous pockets created on the back of

mice. The relatively sparse and distinct vascular network (Fig. 5a) reduced the chance of damaging the blood vessels solely due to injection4. We then injected Evans blue dye (EBD) into the tail vein and sacriced the animals. We observed that TiO2NM at a localized dose of 8 mg kg 1 mouse mass was sufcient to cause increased EBD extravasation at the subcutaneous vasculature on the back of mice (Fig. 5b). These results indicate that TiO2NM can cause vascular leakiness in a localized delivery.

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Figure 5 | TiO2NM promote leakiness of subcutaneous blood vessel. (a) TiO2NM were carefully injected into subcutaneous pockets on the back of mice. EBD was injected into circulation via tail-vein injection. (b) EBD extravasation was more pronounced in the TiO2NM group (8 mg kg 1 dose equivalent to 100 mM). Quantication of EBD showed more endothelial leakiness in the TiO2NM group compared with the untreated mice group. Scale bar, 1 cm. Results represent for meanss.d. from three independent experiments, Students t-test, *Po0.05 compared with untreated control.

TiO2NM cause ECL in a mouse lung metastasis model. Pathological ECL can enhance tumour cell metastasis4. The tumour vasculature is often leaky, which increased nutrients ux to tumours. Leaky vasculature at secondary sites from the initial tumour sites, like distal organs, may act as exit points for metastatic circulating tumour cells, allowing secondary cancer colonization. Studies using inhalation and transdermal delivery of TiO2NM resulted in accumulation in major organs like the liver, kidney and lungs21,22. We therefore reasoned that if accumulated TiO2NM in lung tissue can cause aberrantly leaky blood vessels in the lung vasculature, we might observe higher metastasis of cancer cells. We used an established mouse model where B16F10 mouse melanoma cells injected into circulation metastasized to the lung and showed increased secondary metastatic colonization when there was increased vascular leakiness4,2325. In an acute (1 week) high-dose TiO2NM (50 and 150 mg TiO2NM per kg mouse mass) exposure mouse model, we observed signicantly more supercial melanoma-to-lung metastasis in the TiO2NM-treated groups in a dose-dependent manner when compared with the control group (Fig. 6). There were clear distinct colonies both on the surface and stroma of the lung lobes (Fig. 6a,b). The metastasis load within the lungs were quantied with quantitative PCR (qPCR) analysis for melanin A, a marker of melanoma cells (Fig. 6c), which showed increased melanoma metastasis to the lung in the TiO2NM-treated groups. Scoring of histologically stained lung sections (Fig. 6d) showed signicant increase of tumour penetration and tumour nodules formation with increasing dose of TiO2NM (Fig. 6d). Percentage of lung section coverage by tumours was also signicantly increased with increasing dose (Fig. 6e).

However, the TiO2NM doses in this acute exposure study were very high and any longer experimentation lapsed into very low animal survival rates, making it difcult to study a more chronic treatment. To study subchronic treatment of TiO2NM, we dropped the concentration by 30-fold to 5 mg kg 1 and injected a single bolus of 500,000 cells at the start of the third week and extended the TiO2NM treatment to the end of 4 weeks. We included TiO2 microparticles (680 nm) as another study group. Results showed the gross appearance of tumour nodules on the surface (Fig. 7a) and stroma of the lungs (Fig. 7b) from the TiO2NM-treated group. qPCR analysis of melanin A in lung samples after TiO2NM treatment showed an increased degree of metastatic colonization as compared with control and the microparticles groups (Fig. 7c). Histologically stained section scoring showed a similar trend of TiO2NM increasing metastasis of melanoma cells (Fig. 7d) as compared with the control and microparticles groups. Percentage of lung section coverage by tumours was also signicantly increased in the group with TiO2NM treatment compared with the control and microparticles groups (Fig. 7e).

The tumours in the subchronic experiment were less numerous and larger (Fig. 7b) compared with the acute case (Fig. 6b) because the bolus of B16F10 metastatic melanoma cells are halved and were given twice as long to colonize and multiply. Overall, these mouse models again recapitulated the ndings of the earlier described in vitro experiments that TiO2NM increases ECL.

DiscussionIn this report, we showed a novel non-receptor-mediated and direct binding mechanism of a dense and rigid NM entering into the adherens junction of endothelial cells and disrupting the interaction of VEcadherin as the trigger for ECL. This is in stark contrast to the better understood receptor-mediated mechanism of inducing ECL through binding of a mediator molecule to its receptor with examples like thrombin with PAR-1; bradykinin with bradykinin receptors b1, 2 and 3; histamine with H1 receptors; VEGF and its receptors, VEGFR-1 and VEGFR-226. Chemical-mediated mechanisms that can trigger ECL include oxidative stress and endotoxin though both these cases require cellular uptake of substances27,28 and ECL is a secondary effect. It is still unknown how the adherens junction is targeted by a nanoparticle in the rst place though we postulate that the negatively charged glycocalyx found on the luminal side of endothelial cells may have repelled the sedimenting negatively charged corona-coated TiO2NM (Supplementary Fig. S1) and coupled together with a high mass density; TiO2NM might have bounced and rolled into the adherens junction and entrapped within the connes of the adherens junction niche.

The current paradigm of how NM cross cell barriers is through a series of steps; (a) crossing the cell membrane, (b) endocytosis,(c) intracellular trafcking, (d) repackaging into exocytotic vesicles and (e) traversing the cell membrane28. What we have found is novel and in stark contrast to this prevailing paradigm. The traversing of the mono endothelial cell layer via the route through the adherens junction logically presents less impedence to the nanoparticle. After leakiness is established, this route between the adherens junction allows the nano-exodus of more NM across the endothelium. Therefore, we coined this effect as nanomaterials induced endothelial leakiness (NanoEL).

Exactly how this disruptive binding of TiO2NM to VEcadherin occurs is unknown. As we have shown that negatively charged TiO2NM (Supplementary Fig. S1) binds directly to VEcadherin, it can either bind directly to the extracellular domains where homophilic interaction occurs29 and disrupts that interaction, or the other domains of VEcadherin not involved in the homophilic interaction or even the Ca2 ions itself. EDTA disrupts homophilically interacting VEcadherin by chelating Ca2 ions4. That may provide clues that negatively

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a Control 150 mg kg1

50 mg kg1

Control

b 50 mg kg1 150 mg kg1

*

c d

e

150

*

5

Relative fold change

(melanin A/L27)

Tumour infilration

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Figure 6 | TiO2NM promote vessel leakiness in a melanoma-lung metastasis model. The mice were pre-injected with 106 mouse melanomacells (B16F10) and then with either TiO2NM suspension 50 or 150 mg kg 1 with repeat injections every other day and animals were sacriced on the seventh day. (a) B16F10 in circulation were able to establish more colonies (green arrowheads) in lungs. There was a dramatic increase in supercial lung metastatic load from 50 to 150 mg TiO2NM per kg mouse mass. Scale bar, 1 cm. (b) Eosin-stained sections of lungs showing melanoma colonies (green arrowheads). Scale bar, 50 mm. (c) qPCR analysis of lung tissues showed signicant increase in melanin A expression with increasing dose of

TiO2NM treatment. Data shown were for meanss.d from triplicate qPCR reactions per individual lungs (n 3) in each group. Students t-test, compared

with untreated control *Po0.05. (d) Quantication of the degree of cancer metastasis through blinded scoring made by ten unbiased evaluators of six randomly selected sections per group (n 3 lungs per group). Data are meanss.d. Students t-test, compared with untreated control *Po0.05. (e) Image

analysis results quantifying percentage coverage of tumour area over lung section area (analysis steps in Supplementary Fig. S5) n 18 per group. Tumour

area occupancy over lung section was found to signicantly increase in a dose dependent manner upon TiO2NM treatment. Students t-test, Po0.05 (*treated versus untreated control, # 150 mg kg 1 group versus 50 mg kg 1.

charged TiO2NM with protein corona (Supplementary Fig. S1) may have bound to positively charged side chains of VEcadherin that rigidity of VEcadherin is compromised30. The loss in rigidity in VEcadherin or even denaturation of VEcadherin31 may have caused the loss in homophilic interaction. Whichever the case is, mechanical perturbation at the VEcadherin molecular scale may actually induce the cascade that leads to leakiness. This notion is supported by a study showing that macroscopic hydrodynamic stress on endothelial cells could bring about phosphorylation of VEcadherin32. We further reinforce the idea that mechanical perturbation may drive a different kinase pathway from ligandreceptor-based induction but still converges to the canonical axis of phosphorylation of VEcadherin. Our observations have opened up the exciting possibility of VEcadherin molecule itself as a direct stress sensor.

We only showed that three NM (primary size range 1525 nm) causes ECL, and more investigation needs to be carried out in determining whether this effect is also observed in other similarly sized nanoparticles of other materials. If the ECL-inducing phenomenon is more universal than what we have showed with three different NM, it is then important to also check whether nanomedicine itself causes ECL. Nanomedicine as anticancer drugs act at the molecular level with promising applications in clinics33. While tumours have leaky blood vessels that have enhanced permeability and retention effects (EPR), circulating nanomedicine intentionally introduced into the

vascular system may also inevitably accumulate unintentionally in other non-tumour sites of major organs. While EPR effect can explain metastasis at the primary site of the tumour, based on our study, NM accumulation at non-tumour sites may lead to enhanced secondary metastasis to those sites through NanoEL effects. We have shown in this study that TiO2NM may have detrimental effects; however, the same properties can also be capitalized in nanomedicine, used in situations where leakiness is therapeutic. Situations like traversing the bloodbrain barrier by inducing vascular leakiness to facilitate a lower dose of drugs to the brain though the nonspecic accumulation issue still needs to be resolved. Overall, determination of this size chargemass density window will equip nano-biotechnologists with rules to engineer NM that either avoid or capitalize on this NanoEL effect.

Our mode of TiO2NM delivery in the mouse experiments is articial because the situations where TiO2NM is intentionally introduced directly into the bloodstream would be very rare. While we want to emphasize that these mouse studies are proof of concept experiments and not indicative of real human situations, we would also argue for the need of such controlled animal experiments so we can understand the risk of NM. There are no published reports on TiO2NM-controlled human exposures without any confounding factors over a long period of time and quantication of TiO2NM accumulation in all the major organs. Mouse studies, however, involving respiratory and dermal introduction of TiO2NM did show a low percentage

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a

b

c

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(melanin A/L27)

*

*

Control

680 nm (micro)

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0 Control Nano Micro

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3

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e

50 n=18

*

40

30

% Lung occupied

by tumour

22.8%

20

10

0.07% 0.16%

0

Figure 7 | Low-dose subchronic TiO2NM treatment promote vessel leakiness. The mice were injected with either 5 mg/kg TiO2-NM, 10 mg/kg TiO2 microparticles and vehicle control; total 7 boluses were given with each given every other day. 500,000 B16F10 melanoma cells injection was at 3rd week. Experiment was terminated at the end of 4 weeks. (a) Supercial images showing a dramatic increase in lung metastasis in the TiO2NM group (23.5 nm)

compared with control and microparticles (680 nm). Scale bar, 1 cm. (b) Eosin-stained sections of lungs of mice. Tumour nodule (Tu) indicated the presence of metastasized melanoma in the lung stroma. Scale bar, 50 mm. (c) qPCR analysis of melanoma tumours in lungs showed signicant increase in melanin A expression with TiO2NM treatment. Data are meanss.d. from three different lungs, *Po0.05 when compared with control, Students t-test.

(d) Assessments made by ten unbiased evaluators of six randomly selected sections per group (n 3 lungs per group). Data are meanss.d. Students t-

test, treated with NM compared with untreated control or treated with microparticles versus control, *Po0.05. (e) Image analysis results quantifying percentage coverage of tumour area over lung section area (analysis steps in Supplementary Fig. S5). n 18 per group. There was signicantly higher area

occupancy of tumours in the lung sections of the TiO2NM-treated animals as compared with the control and TiO2 microparticle-treated groups, Students t-test, Po0.05 (*treated versus untreated control).

(lung accumulation estimate of 0.003%) of traversing the various barriers to reach and accumulate in the major organs21,22. Occupational inhalation of manufactured TiO2NM and its corresponding accumulation in the lungs is high with a comparatively low clearance rate34. Deducing from these studies, one can surmise that if given enough exposure time, dose and frequency, it is possible to reach doses that may cause NanoEL. In addition, the pre-existing diseased conditions that increase vascular permeability, like infection35, may exacerbate the onset of NanoEL effect. We, however, need to point out that in order to reach those doses used in our study, we would require extreme circumstances of long-term chronic exposures across possibly damaged epithelia36.

In conclusion, we have described a novel observation that TiO2NM causes ECL and showed that the mechanism preceded oxidative stress. Our data suggest and support a novel mechanism that TiO2NM being small enough to migrate into the inter-endothelial adherens junction niche, binds directly to VEcadherin and disrupts these cellcell interactions. This disruption leads to the phosphorylation of intracellular VEcadherin, which in turn resulted in loss of interaction between intracellular domains of VEcadherin with b-catenin and with p120, triggering the unintended avalanche of actin-rearrangement pathway, which eventually brought about

ECL (mechanism summarized in Fig. 8). The fate of VEcadherin appears to follow the canonical ligandreceptor-mediated pathway where VEcadherin may be internalized and degraded. We have also demonstrated that TiO2NM may cause NanoEL effects resulting in enhanced circulating melanoma metastasis to the lungs. In addition, this study emphasizes the importance of understanding NM interactions with biological systems and especially extracellular niches.

Methods

Functionalization of TiO2NM. FITC was covalently tagged to TiO2NM P25 (Evonik Degussa, USA). Briey, 100 mg of TiO2NM were dispersed in 100 ml of anhydrous ethanol (Fisher Scientic, USA); thereafter 5 ml of aminopropyltriethoxysilane (APTES, Sigma Aldrich, USA) was added to the suspension and stirred at 80 C for 3 h. Then, 25 mg of FITC was added to the mixture and was stirred for another 16 h at 80 C. The FITC-tagged TiO2 was collected, and thoroughly washed with ethanol and subsequently with ultrapure water.

To conjugate mouse IgG to TiO2NM, 10 mg of silanized TiO2NM (after treatment with APTES) was activated with 1.5 ml of 8% glutaraldehyde (Sigma Aldrich) for 6 h in room temperature. Thereafter, the activated TiO2NM were collected, washed thrice with PBS and resuspended in 4 ml of PBS. One hundred and sixty microlitre of suspension (consisting of 400 mg of activated TiO2NM)

were added to 1 ml of PBS solution mouse IgG (20 mg; Millipore, USA). The mixture then was gently agitated for 16 h in 4 C. Following the conjugation process, the mouse IgG-conjugated TiO2NM were collected and washed thrice with PBS. The mouse IgG-conjugated TiO2NM were resuspended in PBS solution and stored in 4 C until further use.

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a

Intracellular Intracellular

Extracellular

-cat

-cat

Before

p120

Y658

Actin

VEcadherin

Y731

~22.5 nm

Classical soluble factors (for example VEGF and histamine)

b

After

Receptor

Src

?

Nanoparticle

P P

Y658

Y731

(1) (2)

(3)

(4)

Endocytic vesicle

Lysosome

Cellretraction>>22.5 nm Phosphorylation Internalization Lysosomal

degradation

Figure 8 | Proposed mechanism of TiO2 NanoEL. (a) The anatomy of an adherens junction. Intact monolayer of connected endothelial cells is maintained by stable VEcadherin homophilic interactions with neighbouring cells. VEcadherin forms a trans-homophillic interaction at the EC domains with another cis-paired VEcadherin complex. b-catenin, p120 and VEcadherin form a complex. Formation of this ternary complex stabilizes the adherens junction38.

Distance of adherens junction is at least 22.5 nm. (b) TiO2NM are small enough to migrate into the adherens junction; they bind and disrupt VEcadherin homophilic interaction (1). This disruption induces the phosphorylation of Y658 of VE-cadherin via a currently unknown kinase pathway, while the Y731 residue is phosphorylated by Src kinase. The phosphorylation at the two residues induces the loss of interaction between VEcadherin and b-catenin and with p120 (2). The loss of interaction of the VEcadherinb-cateninp120 complex destabilizes actin and lead to actin remodelling (3). As a result the cell retracts and leakiness occurs (4). After the binding of TiO2-NM to VEcadherin, VEcadherin might be internalized and further degraded by lysosomes. Fate of VEcadherin: phosphorylation of VEcadherin due to NanoEL may result in internalization and lysosomal degradation. This minimizes the overall amounts of VEcadherin near the vicinity of the cell membrane. TiO2NM might be internalized alongside VEcadherin as it remained bound to

VEcadherin but the nal fate of the TiO2NM is uncertain.

Proximity ligation assay. PLA semiquantication was done by introducing mouse IgG-conjugated TiO2NM to a monolayer HMVEC culture. Conjugation scheme is found in Supplementary Fig. S4. Following treatment, the cells were washed thrice with PBS and xed with 4% paraformaldehyde. Cell cytoplasm were visualized with Alexa 488 Actin staining (1:40; Invitrogen, USA) and the detection of PLA signal was done as per supplier instructions (Olink Bioscience, Sweden). The PLA signals were quantied with Duolink ImageTool software (Olink Bioscience) and normalized against the cell number (n 25).

Permeability transwell assay. The degree of ECL was measured in Transwell insert (with polycarbonate lter, 0.4 mm pore; Corning Costar, Cambridge, MA) as described earlier37. Briey, HMVEC were cultured with density of 20,000 cells per well for 2 days to achieve a conuent monolayer. Thereafter, FITC-dextran (1 mg ml 1, average MW 40,000; Sigma Aldrich, USA) along with NM or

microparticles suspension were added to cells.. At the end of treatment time(30 min), 100 ml samples were taken from the lower compartment and the uorescence signal was quantied with a microplate reader at excitation/emission wavelength of 492/520 nm. Treatment of 2.5 mM EDTA was introduced as positive control.

Immunouorescence staining. Cell junctions were visualized by immunouorescence staining of VEcadherin (Cell Signaling Technology, USA). HMVEC were grown overnight on 8-well chamber slides at initial seeding density of4 104 cells per cm2. The cells were treated by replacing the growth medium with

medium containing TiO2NM. Following the treatment at various time points, media were removed and cells were gently washed with PBS. Thereafter, cells were xed with 4% paraformaldehyde for 15 min, permeabilized using 0.2% Triton X-100 for 15 min and blocked for 1 h with 2% bovine serum albumin (BSA), 0.1%

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Triton X-100 in PBS. Fixed cells were incubated overnight at 4 C with anti VE-cadherin antibody (1:200; Cell Signaling Technology) in 0.2% BSA. Thereafter, the cells were washed thrice with PBS, and incubated for 1 h at room temperature with Alexa 488-chicken anti rabbit antibody (1:400; Invitrogen). Finally, the labelled slides were mounted using ProLong Gold antifade reagent with 40,6-diamidino-2-phenylindole (Invitrogen). Images were obtained using Nikon A1 confocal microscope (Nikon, Japan).

VEcadherin internalization assay was performed in accordance to previous report39. Briey, conuent HMVECs were serum starved for 6 h. At 3 h into the serum starvation, chloroquine (150 mM) was added to prevent lysosomal degradation of the internalized VEcadherin. Cells were subsequently incubated with VEcadherin BV6 (1:100; Millipore, 50 mg ml 1) antibody in medium containing 3% fetal bovine serum at 4 C for 1 h. VEcadherin BV6 antibody specically recognizes the extracellular domain of VEcadherin. Cells were then rinsed with ice-cold serum-free medium to remove excess unbound antibodies and then treated with the various stimuli for 30 min at 37 C. Cells were washed with mild acidic buffer (pH 2.7) containing 3% BSA and 25 mm glycine in order to remove surface-bound VEcadherin. Thereafter, the cells were washed and processed for immunouorescence staining.

TiO2NM internalization study. HMVEC were grown overnight either on 8-well chamber slides or 24-well plates at a seeding concentration of 4 104 cells per cm2.

The inhibition of TiO2NM endocytosis was done by pretreating the monolayer endothelial cells for 1 h with either 10 mM of monodansylcadaverine (MDC; Sigma Aldrich) or 5 mM methyl-b-cyclodextrin (MbCD; Sigma Aldrich) before TiO2

NM treatment. In order to measure the TiO2NM uptake after endocytosis pathway is blocked, the cells were treated with FITC-TiO2 NM (50 and 250 mM) for different time periods (30, 60 and 120 min). Subsequently, the cells were washed and lysed and the FITC signal was measured as mentioned before. In order to study the leakiness after endocytosis pathway is blocked, the monolayer HMVEC is treated with TiO2 NM (50 and 250 mM) for 30 min. Later, the cells were washed and xed with 4% paraformaldehyde before immunostaining as mentioned above.

Separation of membrane-bound and cytosolic FITCTiO2. HMVEC were grown overnight on 6-cm culture dishes. FITCTiO2NM in EndoGRO complete medium were introduced to the cells and media was removed after varioustime points. For the control plate, FITCTiO2NM were added to the cells and immediately removed. After removal of media, cells were immediately washed thrice with PBS and detached from the culture dish. 106 cells were collected for further processing. Membrane and cytosolic fractions were separated by Mem-PER Eukaryotic Membrane Protein Extraction Kit (Thermo Scientic, USA). The FITC signals in the separated fractions were measured at excitation/emission wavelengths of 488/520 nm using Tecan Innite 200 microplate reader (Tecan Inc., Switzerland).

ROCK inhibition assay. HMVEC seeded at an initial seeding density of 4

104 cells per cm2 either in 8-well chamber slides or 24-well transwells and grown till conuent in EndoGRO complete medium. Conuent HMVEC were then rinsed three times with PBS and serum starved for 6 h at 37 C, 5% CO2, following which cells were pretreated with Y-27632 ROCK inhibitor (5 and 10 mM) for 1 h before the addition of TiO2NM. At 30 min post TiO2NM treatment, cells were either processed for immunostaining or subjected to transwell assay.

In vivo subcutaneous vascular leakiness assay. Experimental protocols were approved by Nanyang Technological University Institutional Animal Care and Use Committee (A-0174AZ). Three-weeks-old BALB/c white mice were anesthetized and injected with either TiO2NM or vehicule buffer (3% BSA in PBS). In order to determine the vascular permeability, Evans blue dye (Sigma-Aldrich, USA) was injected into the tail vein. Injection sites were randomly chosen to avoid biasness. The mice were sacriced after 15 min and thereafter the skin was excised and the extravasated dye was extracted. Finally, the amount was quantied by measuring absorbance at 624 nm with microplate reader (Tecan Inc.). The absorbance measured was normalized to the weight of the skin excised.

In vivo murine melanoma-lung metastasis model. For acute study, wild-type C57BL/6J mice were intravenously injected with 106 cells melanoma (B16F10) cells (ATCC: CRL-6475). The mice were treated either with vehicle control in 1% BSA in PBS or with TiO2-NM suspended in 1% BSA in PBS (50, 150 mg kg 1).

NM samples were freshly prepared and were intravenously injected into each mouse every 2 days from Day 0. Mice were sacriced and the lungs were collected for further analysis.

For subchronic study, wild-type C57BL/6J mice were divided into three groups of treatment. One group received 1% BSA in PBS (vehicle control), another received 5 mg kg 1 TiO2-NM and the last group received 10 mg kg 1 of TiO2 microparticles. All TiO2 materials were freshly prepared before intravenous injection. Five hundred thousand murine melanoma cells were intravenously injected.

Total cellular RNA was extracted from the lung samples using the RNeasy brous tissue mini kit (Qiagen, USA) according to the recommended protocol. The

extracted RNA was reverse transcribed to complementary DNA using RevertAid H-Minus First Strand cDNA Synthesis kit (Fermentas, USA), and the metastasis burden was quantied with qPCR of melanin A. The qPCR reactions were performed on ABI 7300 Prism system (Applied Biosystem, USA) using the SYBR Fast Universal qPCR kit (KAPA Biosystems, USA).

Lung samples were xed with 4% paraformaldehyde and stored at 80 C with

O.C.T. Tissue Freezing Compound (Leica Microsystems, USA). Fixed lung samples were cryosectioned at a thickness of 10 mm (Leica Microsystems CM1850, USA).

The sections were stained with eosin (Sigma-Aldrich). The sections were then blind-scored by ten independent assessors. The degree of melanoma inltration was scored with 5 and 0 as the highest and lowest, respectively.

Statistical analysis. Statistical signicance was ascertained through Students t-test with SPSS software (SPPS16.0). P-value of o0.05 is considered signicant.

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Acknowledgements

We acknowledge NUS Academic Research Fund Tier 1 (R-279-000-350-112 to D.T.L.) for funding support. We thank Professors Alison Elder, Robert Hurt, Rouwen Ge,Dr Luigi Calzolai and the late Dr Barry Peter Pereira for helpful discussions.

Author contributions

D.T.L. conceived the hypotheses. D.T.L., M.I.S. and C.Y.T. designed the experiments. M.I.S., C.Y.T., S.L.C., S.L.G., W.F., M.J.N. and H.C.C. performed the experiments. D.T.L., M.I.S., C.Y.T., S.L.C., S.M.T., K.W.N., J.P.X., C.N.O. and N.S.T. analysed the data. D.T.L., S.C.J.L. and N.S.T. contributed the materials. D.T.L., C.Y.T. and M.I.S. wrote the manuscript.

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How to cite this article: Setyawati M.I. et al. Titanium dioxide nanomaterials cause endothelial cell leakiness by disrupting the homophilic interaction of VE-cadherin. Nat. Commun. 4:1673 doi: 10.1038/ncomms2655 (2013).

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