The basal medium Roswell Park Memorial Institute (RPMI) 1640, antibiotic-antimycotic solution, human serum albumin (HSA) (fraction V, fatty acid free), bovine serum albumin (BSA), Hoechst 33258 dye, sodium fluorescein, and UCB were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Non-essential amino acids (NEAA), sodium pyruvate, L-glutamine, fetal bovine serum (FBS), and minimum essential medium (MEM) vitamins were from Biochrom AG (Berlin, Germany). Nuserum IV and rat-tail collagen I were acquired from BD Biosciences (Erembodegem, Belgium). Rabbit anti-β-catenin, mouse anti-ZO-1, rabbit anti-tricellulin, Alexa Fluor 488 goat anti-rabbit IgG, and Alexa Fluor 594 goat anti-mouse IgG were purchased from Invitrogen (Carlsbad, CA, USA). Rabbit anti-caveolin-1, rabbit anti-microtubule-associated protein 1 light chain-3 (LC3), LumiGLO, and cell lysis buffer were acquired from Cell Signaling (Beverly, MA, USA). Horseradish peroxidase-labeled goat anti-rabbit IgG, mouse anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH), rabbit anti-VEGF, and mouse anti-VEGFR-2 were acquired from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Rabbit anti-lamin B1 was from Abcam (Cambridge, UK). Pefabloc® SC AEBSF was obtained from Roche Applied Science (Mannheim, Germany). Horseradish peroxidase-labeled goat anti-mouse IgG, nitrocellulose membrane and Hyperfilm ECL were from Amersham Biosciences (Piscataway, NJ, USA). All other chemicals were of analytical grade and were purchased from Merck (Darmstadt, Germany).

We have previously used an HBMEC line as a simplified model of the human BBB to evaluate the effects of UCB on cell death, cytokine mRNA expression and release, as well as on nitrosative stress (Palmela et al., 2011). This cell line was derived from primary cultures of HBMEC transfected with SV40 large T antigen (Stins et al., 2001) and was cultured in RPMI medium supplemented with 10% FBS, 10% NuSerum IV, 1% NEAA, 1% MEM vitamins, 1 mM sodium pyruvate, 2 mM L-glutamine, and 1% antibiotic-antimycotic solution. For immunocytochemistry, electron microscopy and Western blot studies, cells were seeded at a density of 5 × 10⁴ cell/mL in collagen I-coated coverslips and plates, respectively, and treated after 2 days in culture. For integrity studies, cells were seeded on collagen I-coated polyester transwell inserts (0.4 μM, Corning Costar Corp., USA) at a density of 8 × 10⁴ cell/insert and treated after 8 days in culture. All experiments were performed at confluence. Endothelial cultures were maintained at 37°C in a humid atmosphere enriched with 5% CO₂.

UCB was purified according to the method of McDonagh and Assisi (1972) and a 10 mM stock solution of UCB was prepared in 0.1 M NaOH and used immediately after preparation. The pH value was restored to 7.4 by addition of equal amounts of 0.1 M HCl, and all the procedures were performed under light protection to avoid photodegradation. Confluent monolayers of the HBMEC line were incubated with 50 or 100 μM UCB, or no addition (control), in the presence of 100 μM HSA, for 4, 24, 48, and 72 h. The incubation medium consisted in the regular medium without FBS and Nuserum to avoid disturbance of the final concentration of albumin in the incubation medium.

In the integrity experiments, endothelial cells were cultured on semipermeable filters with two well-defined compartments: the apical or upper compartment, which can be considered as the “blood-side” where UCB and HSA were added, and the basolateral or lower compartment, which is considered the “brain side.” At the end of each incubation period, and after TEER measurement, the cultured inserts were used for permeability studies and media from the upper and lower chambers were collected for Bf measurements. In separate experiments, the cells from the UCB-incubated inserts were collected for UCB extraction with chloroform and the medium of the upper chamber was collected for evaluation of the number of detached cells.

Among the methods used to measure Bf, the peroxidase method is considered practical and reliable, and has been extensively employed (Roca et al., 2006; Ahlfors et al., 2009). Thus, in the present study, we used this method to evaluate Bf levels in our two UCB/HSA molar ratio conditions (Roca et al., 2006).

After determining the starting Bf levels in the two UCB conditions, we monitored Bf levels in the media of the upper and lower chambers of the treated inserts (4–72 h). In order to evaluate the intracellular content of UCB, a chloroform extraction was accomplished based on the study of Brito et al. (2000). Briefly, after lysis of the HBMEC monolayer, each sample was mixed with equal volumes of chloroform. After vortex shaking and centrifugation, UCB concentration in the chloroform extract was determined by direct measurement of the absorption at 454 nm. Results were expressed as nmol of UCB per mg of protein.

TEER evaluation was performed using an EndOhm™ chamber coupled to an EVOMX resistance meter (World Precision Instruments, Inc., USA). Readings were collected before UCB addition (time 0) and after 4, 24, 48, and 72 h of exposure. Results are shown as percentage of variation from average control readings, after deducting the empty insert values.

In order to evaluate the selective paracellular permeability of the HBMEC monolayer after UCB exposure, a permeability assay was conducted with sodium fluorescein (SF) (molecular weight: 376 Da). The permeability was determined as described by Veszelka et al. (2007). Briefly, cell culture inserts were transferred to 12-well plates containing Ringer–Hepes solution (118 mM NaCl, 4.8 mM KCl, 2.5 mM CaCl₂, 1.2 mM MgSO₄, 5.5 mM D-glucose, 20 mM Hepes, pH 7.4) in the basolateral compartments. The SF solution (10 mg/mL SF in 1% BSA solution in Ringer–Hepes) was added to the upper chambers. The inserts were transferred to new wells at 20, 40, and 60 min. Lower chamber solutions were collected to determine fluorescein levels (Hitachi F-2000 fluorescence spectrophotometer, excitation: 440 nm and emission: 525 nm). Flux across cell-free inserts was also measured. The endothelial permeability coefficient P_e was calculated as described by Deli et al. (2005).

At the end of the 72 h period of incubation, the number of viable and non-viable cells in suspension in the upper chamber of the UCB-treated inserts was measured. After media centrifugation, the cell pellet was recovered in RPMI medium and cells were counted with Trypan blue dye exclusion test. Results were shown as number of viable and non-viable cells per insert.

Immunostaining studies were performed to evaluate UCB effects on several relevant proteins that direct or indirectly affect the endothelial barrier functions. The proteins studied included the TJ protein ZO-1, the AJ protein β-catenin, the caveolae-mediated transcytosis protein caveolin-1, as well as VEGF and its receptor VEGFR-2. Detection of these proteins was performed using the primary antibodies mouse anti-ZO-1, rabbit anti-β-catenin, rabbit anti-caveolin-1, rabbit anti-VEGF and mouse anti-VEGFR-2 (all dilutions 1:100), and the secondary antibodies Alexa Fluor 488 goat anti-rabbit and Alexa Fluor 594 goat anti-mouse (1:500). Fluorescence was visualized using a DFC 490 camera (Leica, Germany) adapted to an AxioScope.A1 microscope (Zeiss, Germany). For total fluorescence analysis, fluorescence of five random microscopic fields was acquired per sample. Evaluation of fluorescence intensity was determined using ImageJ 1.29x software (N.I.H., USA) and results were expressed as fold change (mean fluorescence per number of cells vs. control samples).

Total and region-specific β-catenin were further evaluated by Western blot. Subcellular fractionation was performed as described by Abcam (Dr. Richard Pattern). Briefly, cells were lysed with fractionation buffer (250 mM sucrose, 20 mM HEPES, 10 mM KCl, 1.5 mM MgCl₂, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, and 1 mM Pefabloc) and passed through a 25 G needle. The nuclear pellet was collected after centrifugation and the supernatant (cytoplasmic and membrane fractions) was then ultracentrifuged (100,000 g) for 1 h. The cytoplasmic fraction (supernatant) was removed and the pellet (membrane fraction) was ressuspended in cell lysis buffer. The efficiency of fractionation was confirmed with the detection of GAPDH enrichment in the cytoplasmic fraction, Lamin B1 in the nuclear fraction and tricellulin in the membrane region. Results were normalized to total protein staining with Amido Black (Aldridge et al., 2008) due to UCB-induced changes in the expression of the membrane proteins. Therefore, in order to ensure consistency of results, we normalized the intensity of β-catenin in all the extracts to total protein staining.

In order to study the HBMEC process of autophagy after UCB exposure, the conversion of LC3-I (free form) to LC3-II (phosphatidylethanolamine-conjugated form) was also evaluated by Western blot in total protein extracts. The Western blot assays were carried out as previously described (Fernandes et al., 2006). Briefly, total or region-specific protein extracts were separated on a 8 or 12% SDS-PAGE (for β-catenin and LC3 detection, respectively). Following electrophoretic transfer onto a nitrocellulose membrane and blocking with 5% milk solution, the blots were incubated with primary antibody overnight at 4°C [rabbit anti-LC3 (1:1000), rabbit anti-β-catenin (1:1000) or mouse anti-β-actin (1:5000)] and with horseradish peroxidase-labeled secondary antibody [anti-mouse (1:5000) or anti-rabbit (1:5000)] for 1 h at room temperature. Protein bands were detected by LumiGLO and visualized by autoradiography with Hyperfilm ECL.

For scanning EM, cells and vesicles released into the culture medium were doubly fixed with 2% glutaraldehyde in 0.1 M phosphate buffer (PB) and 1% osmium tetroxide in 0.1 M PB, and dehydrated with a graded series of ethanol. Samples were then critical-point dried, sputter coated, and observed by a JEOL JSM-5800LV scanning electron microscope (Tokyo, Japan) at an acceleration voltage of 15 kV. For transmission EM, cells were prepared as described above and then embedded in epoxy resin. Ultrathin sections were stained with uranyl acetate and lead citrate and observed with a Hitachi H-7500 transmission electron microscope (Tokyo, Japan) at an acceleration voltage of 80 kV. For freeze-fracture EM cells, fixed as described, were immersed overnight in 30% glycerol in 0.1 M PB and then frozen in liquid nitrogen. Frozen samples were fractured at −100°C and platinum-shadowed unidirectionally at an angle of 45° in BAL-TEC BAF060 Freeze Etching System. Samples were picked up on formovar-filmed grids and examined with a Hitachi H-7500 electron microscope at 100 kV.

Results are expressed as means ± SEM from, at least, three separate experiments. Significant differences between groups were determined by the two-tailed t-test performed on the basis of equal and unequal variance as appropriate. Statistical significance was considered when P values were lower than 0.05.

We began our studies by evaluating the effect of UCB on caveolin-1, VEGF and VEGFR-2 levels, since these proteins are important players in the mechanisms that regulate endothelial permeability.

As shown in Figure 1, the exposure of HBMEC to both UCB concentrations led to an initial increase in the immunostaining of caveolin-1 that returned to near control levels afterwards (Figure 1A). Maximum fluorescence was observed at 4 h, as represented in Figure 1B (increase of 56 and 91% for UCB 50 and 100 μM, respectively, P < 0.05). In accordance, transmission EM analysis of HBMEC also at 4 h (Figure 1C) evidenced an increased number of caveolae in the UCB-treated cells.

Analysis of VEGF and VEGFR-2 fluorescence showed a surprising pattern of immunostainnings. The cytokine appeared in the perinuclear region, before secretion to the cultured medium as previously observed by us after prolonged UCB exposure (Palmela et al., 2011). On the other hand, receptor staining evidenced to not be restrained to the cell membrane but distributed in the entire cell, probably as a consequence of the interaction between VEGFR-2 and caveolae (Labrecque et al., 2003). Interestingly, the fluorescence showed a progressive increase from 4 to 24 h of exposure, decreasing to control levels afterwards (Figures 2A,B, respectively). Maximum immunostaining was observed at 24 h for both proteins (Figure 2C), correspondent to an increase of ∼16 and 12% in VEGF and VEGFR-2 levels, respectively (P < 0.01).

As shown in Figure 3, the scanning EM analysis revealed that during the early response to UCB (4 h incubation), a great number of vesicles with different sizes were released from the cells treated with both UCB concentrations. Interestingly, the highest concentration produced a very significant release of small vesicles, a distinct pattern from the one triggered by the lowest concentration of UCB. Despite this observation, no alterations were produced in the barrier properties of the monolayer or in the levels of β-catenin and ZO-1 at this time point (results not shown).

Next, we evaluated the effect of UCB in proteins from the TJ and AJ, ZO-1, and β-catenin, respectively, as they are key players in the maintenance of barrier properties of endothelial cells of the BBB.

Since β-catenin has also a crucial role as a transcription factor in the Wnt/β-catenin signaling pathway (Liebner and Plate, 2010), its location is not only relevant in the membrane but also in other subcellular regions. Therefore, we investigated if the cellular distribution of β-catenin was disturbed by UCB. To evaluate this, β-catenin levels were analyzed in three different subcellular locations: nucleus, cytoplasm and plasma membrane. As depicted in Figure 4A, the 24 h exposure to UCB caused a slight elevation of total β-catenin with increases of ∼5% for both UCB concentrations (P < 0.05). Interestingly, and specially for the highest UCB concentration, this minor elevation resulted from a balance between the upregulation of the nuclear fraction (increase of 11%, P < 0.01) and a down-regulation of the membrane content (decrease of 15%, P < 0.01). This pattern of results was also obtained for the 48 h period of incubation (Figure 4B), where the effects to the β-catenin membrane fraction were more marked between UCB concentrations (decrease of 7%, P < 0.05 and 18%, P < 0.01 for UCB 50, and 100 μM, respectively). After 72 h of exposure to the highest concentration of UCB the minor elevation of nuclear β-catenin, along with the sustained decrease in the membrane fraction, produced a significant decrease in total protein (11%, P < 0.05) (Figure 4C).

ZO-1 immunostaining analysis (Figure 5A) revealed also a decrease, although only evident after a 48 h interaction (Figure 5B). This 26% reduction for UCB 50 μM (P < 0.01) and 22% for UCB 100 μM (P < 0.05) suggests a weakness of the intercellular junctions. This alteration was corroborated either by freeze-fracture analysis evidencing loss of TJ strands by both UCB concentrations (Figure 5C), or scanning EM analysis also revealing increased number of retracted endothelial cells with deficient cell-to-cell contacts in the UCB-treated samples (Figure 5D).

TEER and permeability are crucial parameters for the study of endothelial and epithelial barriers, reflecting the quality of the in vitro model and its paracellular barrier characteristics (Wilhelm et al., 2011). By measuring these parameters we were able to evaluate the consequences of UCB disturbance on an otherwise tight barrier.

Figure 6A shows that exposure of the HBMEC monolayer to 100 μM UCB led to a marked reduction of the TEER which, though initiated after 24 h of exposure, attained a maximum 34% decrease at 72 h incubation (P < 0.01). In accordance, the analysis of the HBMEC permeability to SF (Figure 6B) indicated a progressive increase of the flux of this paracellular tracer along the incubation time for the 100 μM UCB, doubling its values at 72 h (P < 0.05).

Autophagy is a highly regulated cellular process that serves to remove damaged proteins and organelles from the cell (Kimmelman, 2011). This complex phenomenon contributes to the maintenance of cellular homeostasis, adaptation to adverse environments and activation of programmed cell death (Codogno and Meijer, 2005; Mizushima, 2007). The induced conversion of LC3-I to LC3-II, the protein associated with the autophagosome membrane, is widely used to monitor autophagy (Kabeya et al., 2000; Tanida et al., 2008). In order to understand if in HBMEC this process is stimulated by UCB treatment, we evaluated the protein levels of LC3 I and II. As depicted in Figure 7, prolonged exposure to UCB enhanced the expression of LC3-II, indicating a higher content of autophagosomes at 72 h (P < 0.01).

Because Bf is believed to be the species crossing the BBB and its quantification is assumed to be a better predictor of BIND risk (Ahlfors, 2001; Ahlfors et al., 2009), we evaluated Bf levels in our current experimental model. The UCB concentrations of 50 and 100 μM (molar ratios of 0.5 and 1.0, respectively) yielded Bf levels of 13.2 and 23.6 nM, respectively.

Our prior results indicated that a prolonged exposure of the HBMEC monolayer to UCB increased the permeability, which could facilitate the passage of UCB across the monolayer. Therefore, we decided to measure the fraction of Bf that passed from the upper chamber of the inserts (where HSA and UCB were added, mimicking the systemic circulation) to the lower chamber (mimicking the brain parenchyma). Values of Bf became significantly detectable only after 72 h incubation, mainly for the highest UCB concentration used (Table 1).

Interestingly, a fraction of UCB remains in HBMEC as revealed after chloroform extraction. The content of UCB in endothelial cells showed a fivefold elevation in cells treated with 100 μM as compared with those exposed to 50 μM for 72 h (Table 1). We next sought to evaluate if the suggested UCB-induced fragility of the monolayer also included the detachment of cells and cellular fragments. As shown in Table 1, 100 μM had the greatest effect on the detachment of cells, increasing the number of cells in suspension, either viable (twofold) or non-viable (12-fold), from both the control condition and the lowest UCB concentration. These results are in agreement with those of TEER and permeability, as well as with those of Bf in the lower compartment and in HBMEC, and yet with the release of vesicles observed by scanning EM, and reflect a fragility of the monolayer that mainly occurs by exposure to the highest UCB concentration in sustained conditions.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.