LDL increases endothelial permeability in an LDLR and cholesterol-dependent way.

<p>(A) HUVECs were plated on top of 0.4 μm pores size transwell inserts and cultured in order to form a confluent and mature monolayer. Cells were either incubated with 100 μg/ml LDL or with the same volume of the control buffer. 24 hours later, cells were washed with serum-free media and the permeability of the monolayer to 70 kDa FITC-dextrans was assessed two hours later, by measurement of the fluorescence at the bottom chamber of the culture system. (B) The same experiment as in (A) with the addition of 2 μg/ml of anti-LDLR or the IgG control at day 7, one hour before the addition of LDL. Fluorescence at the bottom chamber was measured 15 minutes upon the addition of the dextrans. (C) The same experiment as in (B) with the addition of 50 μg/ml of nystatin or the same volume of vehicle at day 8, one hour before the addition of LDL. All the data, except from panel (A), which is a representation of an experiment performed twice with similar results, represent the averages ± standard deviation of at least three independent experiments. Significance values have been calculated using a two-tailed unpaired student <i>t</i> test at the 95% confidence interval (* <i>P</i><0.05).</p

LDL favors the transcytosis of high molecular weight dextrans.

<p>The same experiment as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163988#pone.0163988.g001" target="_blank">Fig 1A</a> was performed. (A) At the end of the experiment, cells were washed twice with PBS and fixed with PFA. A confocal image showing dextrans inside cells is presented. (B) The number of dextran-containing vesicles per cell, two hours after washing from control and LDL conditions was assessed by widefield fluorescence microscopy. Representative images of each condition are shown and the chart represents the quantification of three independent blind experiments in which 50 cells were analyzed. (C) The same experiment as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163988#pone.0163988.g001" target="_blank">Fig 1A</a> with addition of 70 kDA dextrans for 15 minutes, followed by three washes and re-incubation with fresh media both at the top and bottom chambers, 15 minutes later a sample from the bottom chamber was collected and analyzed. (D) The same experiment as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163988#pone.0163988.g001" target="_blank">Fig 1A</a> with the addition of either vehicle or 5 μg/ml of Brefeldin A (BFA). Significance values have been calculated using a two-tailed unpaired student <i>t</i> test at the 95% confidence interval (* <i>P</i><0.05).</p

Lipopolysaccharide (LPS) and unconjugated bilirubin (UCB) modify the distribution of β-catenin in brain endothelial cells.

<p>Cells in mono-culture or co-cultured with astrocytes were fixed and immunostained with an antibody against β-catenin to evaluate its cellular localization (scale bars, 40 and 20 µm, respectively). Disruption of the monolayer with gaps between endothelial cells (*), alterations in protein patterns (arrowheads) with the presence of dot-like staining (yellow arrow), and perinuclear distribution (arrows) are indicated. Representative results from one of two independent experiments are shown.</p

Lipopolysaccharide (LPS) and unconjugated bilirubin (UCB) activate metalloproteinase-9 (MMP-9) and metalloproteinase-2 (MMP-2) released by brain microvascular endothelial cells.

<p>A representative gel from one experiment is shown, where MMP-2 and MMP-9 were identified by their apparent molecular mass of 67 and 92 kDa, respectively (A). The intensity of the bands was quantified by scanning densitometry and results for MMP-9 (B) and MMP-2 (C) were standardized with respect to total protein content. Results are mean ± S.E.M. from at least four independent experiments performed in duplicate. *<i>P</i><0.05 and **<i>P</i><0.01 <i>vs.</i> respective control; ^##<i>P</i><0.01 from 4 h.</p

Lipopolysaccharide (LPS) and unconjugated bilirubin (UCB) disrupt the endothelial monolayer.

<p>Permeability to sodium fluorescein (Na-F_Pe) (A) and transendothelial electrical resistance (TEER) (B) were determined after exposure. All values presented are means ± S.E.M. from at least four independent experiments performed in duplicate. *<i>P<</i>0.05, **<i>P<</i>0.01 and ***<i>P<</i>0.001 <i>vs.</i> respective control; ^§<i>P<</i>0.05 <i>vs.</i> LPS at the same time-point;^#<i>P</i><0.05 from 4 h.</p

Lipopolysaccharide (LPS) and unconjugated bilirubin (UCB) induce cell death in brain microvascular endothelial cells.

<p>Culture medium was collected for determination of lactate dehydrogenase (LDH) activity (A). Nuclei were stained with Hoechst 33258 dye and morphological features of apoptosis are pointed (arrows) (B). The number of apoptotic nuclei was counted and results were expressed as percentage of the total number of nuclei (C). Cell lysates were obtained for caspase-3 activity determination (D). Results are mean ± S.E.M. from at least five independent experiments performed in duplicate. Scale bar, 20 µm. *<i>P<</i>0.05, **<i>P<</i>0.01 and ***<i>P<</i>0.001 <i>vs.</i> respective control; ^§<i>P<</i>0.05, ^§§<i>P<</i>0.01 <i>vs.</i> LPS at the same time-point; ^#<i>P</i><0.05 and ^##<i>P</i><0.01 from 4 h.</p

Lipopolysaccharide (LPS) and unconjugated bilirubin (UCB) alter expression of claudin-5 in brain endothelial cells.

<p>Cells, either in mono-culture or co-cultured with astrocytes, were fixed and immunostained with an antibody against claudin-5 to evaluate its cellular localization, pattern of expression and integrity of the monolayer. Disruption of the monolayer with gaps between endothelial cells (*) and alterations in protein patterns (arrowheads) are indicated. Representative results from one of two independent experiments are shown. Scale bar, 20 µm.</p

Effects of lipopolysaccharide (LPS) and unconjugated bilirubin (UCB) on endothelial integrity in mono-cultures and co-cultures.

<p>Permeability to sodium fluorescein (Na-F_Pe) (A, B) and transendothelial electrical resistance (TEER) (C, D) were determined in mono-cultures (A,C) and co-cultures (B,D) after 24 h exposure. All values presented are means ± S.E.M. from at least two independent experiments performed in triplicate. **<i>P<</i>0.01 and ***<i>P<</i>0.001 <i>vs.</i> respective control; ^§§<i>P<</i>0.01 and ^§§§<i>P<</i>0.001 <i>vs.</i> LPS at the same time-point.</p

Lipopolysaccharide (LPS) and unconjugated bilirubin (UCB) disrupt ultrastructure of brain endothelial cells.

<p>Cells were treated with no addition (control) (A,D,E), LPS (B,F,G) or UCB (C,H,I) and were analyzed by transmission electron microscopy. Black arrows, tight intercellular junctions; grey arrows, disruption of the plasma membrane; arrowheads, invaginations of the plasma membrane; a, apoptotic cells bodies; m, mitochondria; N, cell nuclei; RER, rough endoplasmic reticulum; v, vacuole. Representative results from one of four independent experiments are shown.</p

Lipopolysaccharide (LPS) and unconjugated bilirubin (UCB) alter <i>zonula occludens</i>-1 (ZO-1) expression in brain endothelial cells.

<p>Cells, either in mono-culture or co-cultured with astrocytes, were fixed and immunostained with an antibody against ZO-1 to evaluate its cellular localization and pattern of expression, as well as integrity of the monolayer. Disruption of the monolayer with gaps between endothelial cells (*), alterations in protein patterns (arrowheads) and perinuclear distribution (arrows) are indicated. Representative results from one of two independent experiments are shown. Scale bar, 20 µm.</p