Influence of acitretin on PBEC viability and barrier properties.

<p>(A) Cell viability and cytotoxicity was measured after treatment with different concentrations of acitretin. Maximum amount of LDH (lysis) was set to 100% for the cytotoxicity assay whereas the cell viability of untreated cells (control) was set to 100%. DMSO was used as solvent control. (B) Impact of acitretin on the expression of tight junction proteins in PBEC grown on filter membranes and co-cultivated with SH-SY5Y cells. Scale bar: 40 µm. (C) Internalization of acitretin into PBEC. PBEC transfected with a luciferase-based retinoic acid response element (RARE)-containing reporter plasmid were treated with 2 µM acitretin for 48 hours and the retinoic acid dependent expression of luciferase was determined by luminescence measurement (three experiments; n = 10; unpaired two-tailed t-test; **: p<0.005).</p

Detection of acitretin transport across the BBB.

<p>(A) After 48 hours the amount of acitretin transported across the brain endothelial cell barrier was measured by HPLC. Various concentrations were used to calculate the permeability coefficient of acitretin (P_app (acitretin)). To ensure the tightness of the barrier during the treatment with acitretin, the permeability coefficient of sodium fluorescein was simultaneously determined (acitretin (P_app (NaFITC_acitretin)). (B) Induction of ADAM10-promoter activity in SH-SY5Y cells by acitretin transported across endothelial cells. SH-SY5Y cells were transiently transfected with an ADAM10 promoter reporter plasmid and co-cultured with PBECs for 48 hrs. Acitretin was applied to the upper compartment of the transwell system. The induction of ADAM10 promoter activity by acitretin was monitored by measurement of luciferase activity and was normalized to protein content of whole cell lysate. As control, filters without PBEC (w/o PBEC) were used (three experiments; n ≥10; One Way Anova; Bonferroni post-test; ***: p<0.001).</p

Impact of brain endothelial cells on SH-SY5Y cell viability, signal transduction and APP metabolism.

<p>(A) Cell viability and caspase 3/7 activity of SH-SY5Y cultured with or without porcine brain endothelial cells. Respective values for SH-SY5Y mono-cultures were set to 100%. (B) GSK3β and P-ERK-1/2 expression level in SH-SY5Y cells. Cells were grown in mono-cultures or co-cultivated with PBECs for 48 hrs. Protein levels were determined by Western blot and obtained values were normalized to expression of GAPDH. Values measured within mono-cultures were set to 100%. (C) ADAM10-dependent APP metabolism in SH-SY5Y cells under co-cultivation. Expression level of ADAM10, APP and APP C-terminal fragments were determined by Western blot. Values were normalized to GAPDH and set in relation to respective SH-SY5Y mono-cultures. Amount of secreted APPs-alpha was examined by a dot blot method with the specific APP N-terminal antibody (6E10). Values obtained for mono-cultured SH-SY5Y cells were set to 100% for all analysis. (three experiments; n≥6; unpaired two-tailed t-test; ns: p>0.05).</p

Characterization of the bio-assay model consisting of brain endothelial cells and SH-SY5Y cells.

<p>(A) Scheme of the developed bio-assay model system. (B) Immunofluorescence staining of different tight junction proteins expressed in PBEC mono- and co-culture with SH-SY5Y cells. Scale bars: 40 µm. (C) Immunofluorescent staining of zonula occludens protein (ZO-1) in hCMEC/D3 in mono-culture and in co-cultivation with SH-SY5Y cells. Scale bars: 20 µm. (D) Cell viability and caspase 3/7 activity of brain endothelial cells cultured with or without SH-SY5Y cells. Values obtained for mono-cultures were set to 100%, data represent mean ± standard deviation of three experiments (n ≥8; One Way Anova; Bonferroni post-test; ns: p>0.05; ***: p<0.001). (E) Western Blot analysis of P-gp expressed in brain endothelial cells in mono- and co-culture with SH-SY5Y. (F) Comparison of TEER and permeability coefficients (P_app) of hCMEC/D3 and PBECs grown with or without SH-SY5Y cells. At day eight SH-SY5Y cells were seeded on the bottom of the 24-well plate. P_app of sodium fluorescein was determined 3 hours and 48 hours after seeding of SH-SY5Y cells. (G) HE-staining of brain endothelial cell monolayers grown on top of the filter membranes. Scale bar: 50 µm. (H) Electron microscopy images (a: SEM; b: TEM) of PBEC grown on top of the filter membranes. Arrows indicate the cell-cell connections. Scale bars: 20 µm (a) and 1 µm (b).</p

Impact of polymer-modified gold nanoparticles on brain endothelial cells: exclusion of endoplasmic reticulum stress as a potential risk factor

<p>A library of polymer-coated gold nanoparticles (AuNPs) differing in size and surface modifications was examined for uptake and induction of cellular stress responses in the endoplasmic reticulum (ER stress) in human brain endothelial cells (hCMEC/D3). ER stress is known to affect the physiology of endothelial cells (ECs) and may lead to inflammation or apoptosis. Thus, even if applied at non-cytotoxic concentrations ER stress caused by nanoparticles should be prevented to reduce the risk of vascular diseases and negative effects on the integrity of barriers (e.g. blood–brain barrier). We exposed hCMEC/D3 to twelve different AuNPs (three sizes: 18, 35, and 65 nm, each with four surface-modifications) for various times and evaluated their effects on cytotoxicity, proinflammatory mediators, barrier functions and factors involved in ER stress. We demonstrated a time-dependent uptake of all AuNPs and no cytotoxicity for up to 72 h of exposure. Exposure to certain AuNPs resulted in a time-dependent increase in the proinflammatory markers IL-8, MCP-1, sVCAM, sICAM. However, none of the AuNPs induced an increase in expression of the chaperones and stress sensor proteins BiP and GRP94, respectively, or the transcription factors ATF4 and ATF6. Furthermore, no upregulation of the UPR stress sensor receptor PERK, no active splicing product of the transcription factor XBP1 and no upregulation of the transcription factor CHOP were detectable. In conclusion, the results of the present study indicate that effects of different-sized gold nanoparticles modified with various polymers were not related to the induction of ER stress in brain microvascular endothelial cells or led to apoptosis.</p

Correlation between FACS analysis of VEGFR-2 and formation of capillary-like structures.

<p>The correlation between (A+B) FACS analysis of VEGFR-2 and the (C+D) formation of capillary-like structures for two different donors in the co-culture assay; (A+C) Donor 1 (9.3% VEGFR-2) and (B+D) donor 3 (0.6% VEGFR-2); CD31 staining (endothelial cells, red)</p

Effect of PDGF-BB and VEGF on HUVEC donor 1.

<p>Addition of PDGF-BB and/or VEGF to (A+B) 10.000 and (C+D) 17.500 cells/well of each cell type in the co-culture assay (HUVEC donor 1); amount of added growth factors: (A+C) 10 ng/ml PDGF-BB and (B+D) 40 ng/ml VEGF +10 ng/ml PDGF-BB; CD31 staining (endothelial cells, red)</p

of capillary-like structures in the co-culture system.

<p>(A) Pre-processing of the obtained mosaix images for analysis in Angioquant, (B) explanation of length, junctions, area and complex as defined in Angioquant, CD31 staining (endothelial cells, red)</p

Co-culture assay with supportive ovine carotid-artery derived cells and HUVECs.

<p>DAPI (all cell nuclei, blue) and CD31 staining (endothelial cells, red)</p

Correlation of VEGFR-2 with VE-Cadherin and capillary-like structures.

<p>(A) Confluent and sparse endothelial seeding in HUVEC/cartid artery-derived cell co-culture systems; CD31 endothelial cell staining; (B) Immunoprecipitated VEGFR2 blotted with anti-phosphotyrosine (PY 20) for detection of phosphorylated levels of VEGFR2; whole cell lysates were blotted with α-Tubulin to show equal loading.</p