Fabrication of tissue engineered vessels
Scaffold-directed human engineered vessels were generated under flow bioreactor conditions as described [17]. Briefly, non-woven polyglycolic-acid meshes (PGA; BMS) were coated with polycoprolactone (PCL, Purac) and polyglycolic acid (PLA, Purac) by dipping into a 1.75% (w/w/w) solution of PCL/PLA/tetrahydrofuran (Sigma Aldrich). A tubular shape (length 10 mm and inner diameter 2 mm) was obtained by heat welding before external coating with a 10% (w/w) PCL/tetrahydrofuran solution. After sterilization with 70% ethanol for 30 min followed by 3 washes with PBS, scaffolds were pre-incubated in advanced DMEM overnight before cell seeding. Human umbilical cord smooth muscle cells were isolated as described [17] and 2 × 106 cells/cm2 were seeded in the inner surface of the vascular scaffold using fibrinogen (10 mg/mL clottable protein, Sigma Aldrich) and thrombin (10 mU) as cell carriers [18]. After a short static incubation period of 3 days, vascular constructs were exposed to dynamic conditioning in a flow bioreactor, where the flow of nutrient medium (Advanced DMEM (Invitrogen) with 10% FBS; 0.05% Penicillin/Streptomycin, 1% L-glutamine and 1.5 mM L-ascorbic acid) was directed through the inner lumen of the bioreactor circulation loop at 6 mL/min. After 14 days of flow conditioning, vascular grafts were endothelialzed with HUVECs (1.5 × 106 cells/cm2) seeded into the lumen followed by cultivation under static conditions for 5 days in complete endothelial growth medium (EBM-2; Lonza) supplemented with 10% foetal bovine serum (FBS) and growth supplements to form EGM complete media FBS. After the static phase, vascular grafts were placed back in the bioreactor for 14 additional days with increasing medium flow (4 to 6 mL/min) in complete EGM-2 with 10% FBS.
Characterization of engineered vascular tissue
For histological characterization, engineered vessels were fixed in formalin (Fisher), dehydrated through a graded ethanol series using a Sakura Tissue Processor (Sakura), embedded in paraffin and sectioned at 7 μm thickness. Sections were deparaffinised, rehydrated through a graded ethanol series and stained using haematoxylin & eosin (SigmaAldrich) following the manufacture’s instructions. For immunofluorescence analyses, tissues were cryopreserved in Cryomatrix (ThermoFisher) and sectioned at 20 μm thickness using a cryotome (Leica). Immunostaining was performed as described [17] using antibodies specific for the EC marker CD31 (clone JC/70A Biolegend, 1:50) and α-smooth muscle actin (clone 1A4 SigmaAldrich, 1:200). Sections were counter-stained with DAPI and imaged with an inverted microscope (Carl Zeiss). Endothelium barrier integrity was analyzed by injecting Evans blue (Sigma Aldrich) at a final concentration of 0.5% in the circulation loop of the bioreactor for 10 min followed by continuous PBS washing for 20 min. Vessels were cut open longitudinally and en face preparations were analysed macroscopically with photo documentation.
Preparation of HDL and PBMCs
All experiments were conducted under an approved clinical protocol (UBC Clinical Ethics Research Board H14–03357). Upon receipt of written informed consent, 100 mL of fasted blood was collected from normolipidemic healthy donors into vacutainer tubes. Plasma HDL (1.063–1.21 g/mL) was isolated by sequential potassium bromide gradient ultracentrifugation as described [19]. The purity of the HDL preparations was verified by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie blue staining to ensure no low-density lipoprotein (LDL) or albumin contamination. Eight independent donors were used across the experiments, 6 isolated in-house and 2 commercially obtained (Leebioscience). Human-derived, lipid free apoA-I was a kind gift from CSL-Behring. Immortalized human THP1 monocytes (ATCC) were cultured in RPMI containing 10% FBS, 1% Pen/Strep, 2 mM L-glutamine and 0.1% β-mercaptoethanol. Primary human PBMC were isolated from healthy donors by centrifugation on a continuous density gradient (Lymphoprep™, Stemcell) following the manufacturer’s instructions. Freshly isolated PBMC were fluorescently labeled with 10 μM of Cell-Tracker Red for 30 min (Invitrogen) following the manufacturer’s recommendations.
Monocyte adhesion in engineered vessels
Vascular grafts were perfused with complete EGM-2 with 2% FBS. 1 μM Aβ42 or Aβ40 monomers were injected directly into the graft chamber to mimic Aβ originating from the brain (antelumen) side of the vessel. At time points ranging from 2 to 72 h, THP1 cells were fluorescently labeled with Cell-Tracker Red as described above, injected in the graft circulation at a concentration of 1 × 106 cells/mL and maintained under flow conditions for 3 h. For HDL experiments, vascular grafts were perfused with luminal HDL (200 μg/mL) for 2 h before injecting Aβ in the antelumen side for 8 h. Tissues were longitudinally cut open, washed extensively with PBS and fixed with 4% PFA. After 20 min, tissues were washed 3 times with PBS and mounted in Prolong Gold antifade reagent with DAPI. For each independently seeded tissue, adherent monocytes and monocytes undergoing diapedesis were counted in 3 random squares of 1.23 mm2 using a z-stack covering the whole tissue thickness with a SP8 confocal microscope (Leica), averaged and expressed as percent of vehicle normalized to 100% for Fig. 1d and f or percent of Aβ normalized to 100% for Fig. 1 h and j, and Fig. 8a and b.
Static monotypic cell culture
hCMEC/D3 (Fisher; passage 27–35), and HUVEC (passage 4–7, isolated as described [17]) cells were cultured using complete EGM-2 with 2% FBS, ECs were cultured in a humidified incubator at 37 °C at 5% carbon dioxide. For mechanistic experiments, ECs were treated with SR-BI blocking antibody (NB440–113 Novus, 1:500), the SR-BI inhibitor block lipid transport-1 (BLT-1, SigmaAldrich, 10 μM), the eNOS inhibitor L–NG-nitroarginne methyl ester (L-NAME, SigmaAldrich, 1 mM), the receptor associated protein (RAP, Oxford biome, 1 μM), the RAGE blocking antibody (176,902, R&D Systems, 1:50), heparin (10 mU), heparinase (SigmaAldrich, 0.2 mM) or the CD36 blocking antibody (JC63.1, ABCAM, 1:500) for 1 h before HDL priming. For miRNA experiments, cells were transiently transfected using Lipofectamine 2000 (Life Technology) 2 h before Aβ stimulation with the miR-223 mimetic (Life Technology, 4,464,066 (MC12301), 100 nM) or miR-223 inhibitor (Life Technology, 4,464,084 (MH12301), 100 nM) in EBM-2 containing 0.2% BSA.
Monolayer PBMC adhesion assay
ECs were seeded at 1 × 105 cells/well in 24-well plates and cultured until confluent for 2 to 3 days. On the day of the assay, ECs were primed for 2 h with 100 μg/mL bovine serum albumin as vehicle control or 100 μg/mL HDL before stimulation with 1 ng/mL of TNF-α (Preprotech) or various concentrations of monomeric Aβ40 and Aβ42 (0.001–1 μM, California Peptide), prepared as described below. After 3 h, 5 × 105 Cell-Tracker Red-labeled PBMC per well were added to ECs for 3 additional hours before washing 3 times with PBS to remove non-adherent PBMC. Cells were then fixed with 4% PFA for 15 min before 3 additional PBS washes and 4′,6-diamidino-2-phenylindole (DAPI) counterstaining. For each independent experiment, adherent monocytes were counted in 5 random squares of 7.84 mm2 using a fluorescent inverted microscope (Zeiss), averaged and expressed as percent of vehicle normalized to 100%. DAPI counterstaining was used to ensure endothelial cell coverage.
Aβ oligomerization/fibrilization and electron microscopy confirmation
Recombinant Aβ40 and Aβ42 peptides (California Peptide) were dissolved in ice-cold hexafluoroisopropanol (HFIP). The HFIP was removed by evaporation overnight. To prepare soluble monomers, the peptide film was reconstituted in DMSO to 5 mM, diluted further to 100 μM in DMEM and used immediately. Oligomers were prepared by diluting the 5 mM DMSO peptide solution in phenol red-free F12 medium (Life Technologies) to a final concentration of 100 μM and incubating for 24 h 4 °C. Fibrils were prepared by diluting the 5 mM peptide solution in 0.1 uM of HCl to a final concentration of 100 μM and incubating for 24 h at 37 °C. Aβ monomer, oligomer and fibril preparations were then either used to stimulate hCMEC/D3 monolayers, as given, or were analysed by Transmission Electron Microscopy (TEM) and dot blot. For TEM, 0.5 μL of 100 μM Aβ preparation was diluted in 2 μL filtered distilled water, spotted onto formvar-coated 200-mesh nickel grids (EM Sciences) and allowed to dry. Grids were then negatively stained with 0.5% aqueous uranyl acetate for 30 s, and viewed on a FEI Tecnai G2 Spirit Transmission Electron Microscope. For dot blot, aliquots of Aβ(0.1 μM) were added to PVDF membrane, which were dried and blocked in 3% skimmed milk PBST. After 1 h, blots were incubated with β-amyloid 1–16 antibody (6E10 Biolegend 1:500), amyloid A11 oligomeric antibody (AB9234 Millipore, 1:1000) or amyloid fibrils OC antibody (AB2286 Millipore, 1:1000) in blocking buffer for 16 h, washed extensively in PBST and incubated with anti-mouse or anti-rabbit (1:1000) secondary antibody in blocking buffer. After 1 h, blots were washed as above and developed using enhanced ECL and a ChemiDoc MP imager.
Measurement of intracellular NO
ECs were seeded at 5 × 105 cells/well in 6-well plates and cultured for 2 days until confluent in complete EBM-2. Sixteen hours before the assay, ECs were serum-depleted in EBM-2 containing 0.2% FBS. On the day of the assay, ECs were incubated with 100 μg/mL of HDL in serum-depleted EBM-2 medium containing 1 μM of 4,5-diaminofluorescein diacetate (DAF2, Caymanchem) at 37 °C. After 6 h, ECs were washed with PBS, trypsinized, and triazolofluorescein fluorescence was measured (excitation wavelength of 485 nm, emission 538 nm), using an Infinite M200Pro plate reader (Tecan). In addition to DAF2 measurement, the phosphorylation of eNOS at Ser1177 was compared to total eNOS by immunoblotting (below) in cell lysates harvested 15 min after HDL treatment.
Cell surface biotinylation
ECs were seeded at 5 × 105 cells/well in 6-well plates and cultured for 2 days until confluent. On the day of the assay, ECs were treated with HDL, Aβ or TNF-α as above. After stimulation, EC monolayers were washed twice with ice cold PBS (pH 8), cooled on ice for 15 min and biotinylated with 250 μg/mL EZ-link™-sulfo-NHS-biotin (ThermoFisher Scientific) in PBS (pH 8) at 4 °C. After 1 h the reaction was stopped with a 5 min incubation in DMEM with 10% FBS. After two additional PBS washes, ECs were lysed in RIPA buffer. Following quantification of protein concentration using a BCA assay (Thermofisher Scientific), at least 100 μg of protein were incubated with streptavidin-conjugated sepharose beads (Pierce) at 4 °C overnight. Beads were washed 3 times with RIPA buffer and the recovered proteins were resolved on a SDS-PAGE.
Monolayer aβ association, binding and uptake
ECs were seeded at 3 × 105 cells/well in 12-well plates and cultured until confluent for 2 to 3 days. On the day of the assay, ECs were primed for 2 h with 100 μg/mL HDL before stimulating with 0.1 μM of Aβ40 and Aβ42 monomers at 37 °C for total association, or at 4 °C for cell surface binding. After 3 h, hCMEC/D3 were washed 3 times with PBC and lysed in RIPA buffer containing 10 mM Tris pH 7.4, 150 mM NaCl, 1.0% NP-40, 1.0````% sodium deoxycholate, 0.1% SDS and cOmplete protease inhibitor with EDTA (Roche). Aβ40 (KHB3442, Life Tech) and Aβ42 (KHB3482, Life Tech) were quantified using commercial ELISAs and normalized to total protein concentration. For Aβ uptake, hCMEC/D3 were seeded at 1 × 105 cells/well in 24-well plates and cultured to confluence for 2 to 3 days. On the day of the assay endothelial, ECs were primed for 2 h with 1 mg/mL of HDL before stimulating with 1 μM monomeric FITC-Aβ40 and FITC-Aβ42 (Bachem) prepared as described above. After 3 h at 37 °C, hCMEC/D3 were washed 3 times with PBS and fixed in 4% PFA for 20 min. After one Tris-HCl and two PBS washes, hCMEC/D3 were mounted in Prolong antifade reagent.
Molecular biology
For mRNA, cells were lysed in Trizol (Invitrogen) and RNA was extracted and treated with DNase I (Invitrogen) according to the manufacturer’s protocol. cDNA was generated using oligo-dT primers and Taqman reverse transcription reagents (Applied Biosystems). Real-time quantitative PCR was done using FastStart Universal SYBR Green Master reagent (Roche) on a Light Cycler 96 system (Roche) to quantify gene expression relative to vehicle using specific primer against ICAM-1 (fwd: ATGGCAACGACTCCTTCTCG; rev: CGCCGGAAAGCTGTAGATGG) and VCAM-1 (fwd: TGTTTGCAGCTTCTCAAGCTTTT; rev: GATGTGGTCCCCTCATTCGT) and normalized to GAPDH (fwd: CCTGCACCACCAACTGCTTA; rev: CATGAGTCCTTCCACGATACCA). MiRNA was isolated using an miRNeasy mini kit (Qiagen) following the manufacturer’s instructions, respectively. Reverse transcription was performed using 2 μg of total RNA with specific miRNA primers (Life Technology). MiR223 and U6 were quantified using specific TaqMan probes (Life Technology).
Immunoblot
ECs were lysed in RIPA buffer containing either cOmplete protease inhibitor or Phosphostop (Roche) and quantified using a BCA assay. Equal amounts of total protein were separated by SDS-PAGE followed by electrophoretic transfer to polyvinylidene fluoride (PVDF) membranes (Millipore). After blocking membranes for 1 h with 5% skim milk powder in PBST, or 5% BSA in TBST for phosphoproteins, ICAM-1, (EP1442Y Abcam, 1:1000), VCAM-1 (EPR5047 Novus, 1:1000), peNOS (ser1177 Cellsignalling, 1:1000), eNOS (M221 ABCAM, 1:1000), AnxA1 (D5V2T, Cellsignalling, 1:1000), p-Akt (Ser473 D9E, Cellsignalling, 1:2000), Akt (9272, Cellsignalling, 1:1000), p-NF-κB p65 (Ser536 93H1, Cellsignalling, 1:1000), NF-κB p65 (D14E12, Cellsignalling, 1:1000), p-SAPK/JNK (Thr183/Tyr185 G9, Cellsignalling, 1:1000), JNK (2C6, Cellsignalling, 1:1000), p-p42/44 MAPK (Erk1/2) (Thr202/Tyr204 D13.14.4E, Cellsignalling, 1:1000), p42/44 MAPK (Erk1/2) (137F5, Cellsignalling, 1:1000), p-Stat3 (Tyr705, Cellsignalling 1:1000), Stat3 (124H6, Cellsignalling, 1:1000) and GAPDH (MAB374 1:10,000, Millipore) were immunodetected by incubating for 16 h in primary antibody in blocking buffers. Membranes were washed extensively with PBST or TBST, and incubated with anti-mouse or anti-rabbit (1:1000–10′000, Jackson ImmunoResearch) secondary antibody in blocking buffer. After 1 h, membranes were washed as above and developed using enhanced chemiluminescence (ECL, Amersham) and a ChemiDoc MP imager (Biorad). Densitometric images were captured with ImageJ and band intensity normalized to GAPDH as a loading control.
Beta-sheet formation assay
Cell-free Thioflavin-T fibrillization assays were performed on an Infinite M2000 Pro plate reader (Tecan) as described (Truran 2015). Briefly, 10 μM monomeric Aβ40 or Aβ42 were incubated in a buffer consisting of 20 mM of Thioflavin-T in 150 mM NaCl and 5 μM of HEPES at pH 7.4, with and without 1 mg/mL of HDL, at 37 °C with 20 s of orbital shaking (3 mm amplitude) every 5 min in a black 96-well plate. Formation of fibrillar β-amyloid pleated sheets over time was monitored by excitation at 440 nm and measuring emission intensity at 490 nm every 5 min up to 12 h in total.
Human brain protein extraction and ELISA
Frozen brain tissues (cortex Brodmann area 9 and cerebellum) were provided by the Harvard Brain Tissue Resource Center under an approved UBC protocol (C04–0595) and extracted with 8 volumes of ice-cold carbonate buffer (100 mM Na2CO3, 50 mM NaCl, pH 11.5) containing cOmplete protease inhibitor (Roche Applied Science) by manual homogenization with a tissue probe. After incubating on ice for 10 min, lysates were clarified by centrifugation at 16,600 rcf for 45 min at 4 °C. The supernatant was removed and neutralized by adding 1.5-volumes of 1 M Tris-HCl pH 6.8 to give a final pH of approximately 7.4. Brain tissues from all samples were extracted in an identical manner, and fractions were aliquoted and immediately frozen at −80 °C until analysis. Protein concentrations were determined using the Lowry Protein Assay (Biorad). ICAM-1 (ab174445, ABCAM), VCAM-1 (ab187393, ABCAM), Aβ40 (KHB3442, Life Tech) and Aβ42 (KHB3482, Life Tech) in carbonate extracts were quantified using commercial ELISAs. ELISA data were normalized to total protein concentration of the extract. For immunofluorescent staining, brains were sectioned at 20 μm, rehydrated in PBS and blocked in 4% paraformaldehyde (PFA). After 20 min, sections were washed once with Tris-HCl (0.5 mM, pH 8), twice with PBS, and blocked in blocking buffer (5% goat serum and 1% BSA). After 60 min, section were incubated at 4 °C with antibodies against CD31 (WM59 Biolegend, 1:50), ICAM-1 (EP1442Y ABCAM 1:200), and VCAM-1 (EPR5047 ABCAM 1:200) in blocking buffer overnight. Sections were washed 3 times in PBS and incubated at RT with Alexa-488 anti-mouse and Alexa-594 anti-rabbit fluorescently labeled secondary antibodies (LifeTechnologies, 1:600). After 45 min and 3 additional PBS washes, sections were mounted in Prolong Gold antifade reagent with DAPI (LifeTechnologies) and imaged on an inverted fluorescent microscope (Zeiss)
Statistical analysis
All statistical analyses were performed using SPSS and p-values <0.05 were considered significant. Data were obtained from at least 3 independent experiments and are presented as mean ± SD if not indicated otherwise. Data were first log transformed and analyzed by two-way ANOVA with a blocking factor (experiment) with direct comparison when comparing two treatments or Dunnett’s or Bonferonni multi-comparison tests. After statistical calculations, vehicle data were normalized to 100% and represented as a dashed line in the graph, and tested conditions were expressed and graphed as percentage of vehicle if not otherwise stated.