Additional file 1 of Cerebrovascular amyloid Angiopathy in bioengineered vessels is reduced by high-density lipoprotein particles enriched in Apolipoprotein E

Additional file 1: Supplemental Figure 1. Schematic view of the bioengineered vessel and bioreactor. The left panel represents a schematic of the bioreactor design with the peristaltic pump, media container, circulation loop, and bioengineered vessel chamber. The bioengineered vessel separates the tissue chamber (brown) where Aβ is injected from the circulation loop (red) where HDL is circulated. The right panel depict the schematic of bioengineered vessels composed of EC (yellow), SMC (orange) without (bipartite) or with (tripartite) astrocyte (blue). Non-woven PGA/PCL scaffold is showed in white and defines the number of SMC layers. Supplementary Figure 2. HDL and mix LDL delay Aβ42 fibrillization. (a) Aβ fibrillization was measured in a Thioflavin T cell-free assay over 7200 min. (b) Maximal fluorescence, (c) time to half-maximal fluorescence (V50) and (d) lag phases were calculated using Boltzmann curve analysis (vehicle R2 = 0.91; HDL R2 = 0.94; LDL R2 = 0.4). Points in graphed data represent individual experiments, bars represent mean, error bars represent ±SEM and data are presented as mean +/− SEM and analysed by one way ANOVA *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001. Supplementary Figure 3. HDL does not reduce deposition of preformed Aβ42 oligomers or fibrils in engineered tissues. (a) 1 μM Aβ42 was injected into the tissue chamber concomitantly with circulation of 200 μg/mL HDL through the lumen before measuring Aβ42 deposition in the GluHCl fraction by ELISA and examining Thioflavin S staining (white) after 24 h (* remaining scaffold). (b) Aβ42 oligomers were prepared by incubating monomers in RPMI for 48 h at 4 °C before injection of 1 μM oligomers into the tissue chamber while circulating 200 μg/mL HDL through the lumen. After 24 h, Aβ was measured in both RIPA and GluHCl fractions by ELISA. (c) Aβ42 fibrils were prepared by incubating monomers in RPMI for 48 h at 37 °C before injection of 1 μM fibres into the tissue chamber while circulating 200 μg/mL HDL through the lumen. After 24 h, Aβ was measured in both RIPA and GluHCl fractions by ELISA. (d) 1 μM Aβ42 monomers were injected into the antelumen 48 h before circulating 200 μg/mL HDL through the lumen. After 24 h, Aβ42 deposition was measured in RIPA and GluHCl fractions by ELISA. Thioflavin-S staining are representative image of 3 individual tissues. Points in graphed data represent individual bioengineered vessels, bars represent mean, error bars represent ±SEM and analysed by Student’s t-test. Supplementary Figure 4. HDL reduces FITC-Aβ42 uptake by EC and HDL does not increase Aβ42 co-localization with lysosome in SMC. EC were grown in 2D monolayers for 2 d before treating with 1 μM FITC-Aβ42 without or with 200 μg/mL HDL for 3 h (a) or 24 h (b) before dissociating cells and counting using flow cytometry. (c) SMC were grown in chamber slides for 2 d before treating with 5 μM FITC-Aβ42 (depicted as yellow) without or with 200 μg/mL HDL. After 24 h, SMC were fixed and stained for the lysosomal marker LAMP1 (magenta) before imaging using confocal microscopy. Points in graphed data represent individual bioengineered vessels, bars represent mean, error bars represent ±SEM and analysed by Student’s t-test *P < 0.05 and **P < 0.01. Fluorescent images are representative of two separate experiments. Supplementary Figure 5. HDL does not alter Aβ42 entry from the circulation into the vascular wall. (a) 1 μM Aβ42 monomers were injecting into the tissue chamber with or without circulating 200 μg/mL of HDL through the lumen. After 24 h tissues were lysed in RIPA and RAGE levels were measured in bioengineered tissues using Western blot and normalized to GAPDH. 1 μM Aβ42 monomers were injecting either into the tissue chamber or the circulation loop with or without circulating 200 μg/mL of HDL through the lumen. After 24 h, Aβ42 was measured in the tissue chamber (b), circulation (c), and tissue lysed in RIPA (d) by ELISA. (e) 1 μM Aβ42 was injected into the tissue chamber with or without a blocking antibody against RAGE in the circulation. After 24 h, tissues were lysed in RIPA Aβ42 levels were measured by ELISA. Points in graphed data represent individual bioengineered vessels, bars represent mean, error bars represent ±SEM and analysed by Student’s t-test or one-way ANOVA *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001. Supplementary Figure 6. Aβ deposition co-localizes with vascular ECM in post mortem human cortex. Human cortex (Brodmann area 9) were stained for Aβ using 6E10 (yellow) and collagen-IV (a), laminin (b), biglycan (c), HSP2 (d) and versican (e) (magenta). Expanded views of a vessel with CAA (1) and cortical Aβ plaques (2) are shown on the right. Images are representative of four individual human donors. Supplementary Figure 7. Aβ42 forms a complex with HDL and reduces HDL’s cholesterol efflux activity. (a) 1 μM Aβ42 were incubated either with 200 μg/mL of HDL or BSA for 24 h at 37 °C before gel-filtration chromatography separation. HDL alone (b) or Aβ42 monomers alone (c) were separated by gel-filtration chromatography and dot blotted fractions were probed against Aβ (6E10) or HDL (apoA-II). Graphs and dot-blots show representative experiments from three individual FPLC runs. RAW 264.7 cells were loaded with H3-cholesterol and cholesterol efflux was measured in the presence of HDL (d), lipid-free apoA-I (e) or no acceptor (f), pre-incubated with or without 1 μM Aβ42 for 24 h. Points in graphed data represent individual experiments, bars represent mean, error bars represent ±SEM and analysed by Student’s t-test *P < 0.05, ***P < 0.001 and ****P < 0.0001.

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