AD individuals and controls
Cerebral VSMCs were isolated from small intracerebral and leptomeningeal arteries (Brodmann’s areas 9/10) from late-stage AD individuals with CAA and non-demented age-matched controls with little or no pathology and without CAA. For clinical and neuropathological characteristics see Supplementary Information, Table S1.
Human VSMC culture
Pial arterial VSMCs were isolated and characterized as reported previously22
. We used primary early passage 1 VSMCs.
Adenoviral sh.SRF and MYOCD gene constructs
gene constructs were as we previously reported22
Adenoviral sh.LRP construct
The LRP RNAi gene construct was cloned into the BLOCK iT U6 RNAi expression entry vector.
Gene transfer to VSMC
This was performed as reported previously22
Cellular clearance of Aβ deposits
The multi-spot glass slides were coated with Cy3-labelled Aβ42 or Aβ40 (ref. 26
) at 5^µg per spot without cells, with control cerebral VSMCs or AD VSMCs, AD VSMCs transduced with Ad.shSRF
, or control VSMCs transduced with Ad.MYOCD
. Cells (2,500 per spot) were incubated for 5 days in DMEM containing 10% heat-inactivated FBS, penicillin and streptomycin (Invitrogen). Cells were labelled with Cell Tracker Green CMFDA (Invitrogen C7025) and Lysotracker Blue DND-22 then fixed with 4% paraformaldehyde. Images were scanned using a Zeiss 510 meta multi-photon/confocal microscope with a 543 nm HeNe laser to detect Cy3-Aβ42, a 488 nm Argon laser to detect Cell Tracker Green and an 800-nm tuned Ti:sapphire Laser (Mai Tai Spectra Physics) to detect Lyostracker Blue. Twenty randomly chosen spots per slide from 3–4 independent cultures per group were analysed. The relative Cy3 signal intensity was measured with the NIH Image J software.
QPCR for LRP mRNA in cultured VSMC
QPCR was performed using SYBR Green (Bio-Rad) and the Stratagene MX3005P QPCR system.
SREBP2 luciferase reporter assay
We cloned from HeLa genomic DNA the human SREBP2 promoter (1,375 bp encompassing one CArG element) and intron 1 sequences (1,315 bp encompassing 2 CArG elements) for responsiveness to SRF/MYOCD. Following sequence validation, we cloned the SREBP2 promoter into the pGL3 basic vector (Promega) either alone or in combination with the intronic sequence. Mutations in the SREBP2 CArG elements were performed with a QuikChange mutagenesis kit (Stratagene) where the first 3 nucleotides of the CArG element were changed to GTC. Luciferase activity was measured using a Dual-Luciferase Reporter Assay System (Promega) and normalized to a control (Renilla) vector.
SREBP2 knockdown in cultured human VSMCs
Control siRNA (siCNTRL, Santa Cruz, sc-37007) or SREBP2 siRNA (sc-36559) were transfected into cultured VSMCs using Metafectene (Biontex) according to the manufacturer’s instruction.
Expression of SRF, MYOCD, SREBP2 and LRP was studied by western blot analysis in human cerebral VSMCs and small cerebral leptomenigeal arteries (Brodmann’s areas 9/10) derived from AD patients with CAA and controls. VSMC samples were washed in cold phosphate buffer saline and lysed with a RIPA buffer. Small cerebral arteries samples were additionally sonicated in the RIPA buffer. Proteins were quantified with a protein assay kit (Pierce).
Immunostaining for nuclear SREBP2 and LRP
AD VSMCs infected with Ad.shGFP or Ad.shSRF or control VSMCs infected with Ad.LacZ or Ad.MYOCD were cultured for 48 h and fixed in 4% paraformaldehyde. Cells and human brain sections (Brodmann’s area 9/10) were then stained using a rabbit anti-human SREBP2 antibody (Abcam, ab30682) and mouse anti-human LRP (Calbiochem, 438192) followed by incubation with secondary antibodies, such as FITC-conjugated goat and rabbit, CY3 conjugated donkey anti-mouse and TO-PRO3, a fluorescent nuclear marker (Invitrogen). Cells and tissue were scanned using a Zeiss 510 meta multi-photon/confocal microscope with a 543-nm HeNe laser to detect Cy3, a 488-nm Argon laser to detect FITC, a 633-nm HeNe laser to detect TO-PRO3, and an 800-nm tuned Ti:sapphire Laser (Mai Tai Spectra Physics) to detect methoxy-X04.
MYOCD luciferase reporter assay
We cloned the mouse Myocd promoter from genomic DNA with or without the conserved hypoxia response element (HRE). Following sequence validation, we cloned the MYOCD promoters into the pGL3 basic vector (Promega). PAC1 VSMC were transfected with luciferase reporters, then incubated in DMEM containing 2% FBS under normoxic (21% oxygen) or hypoxic (1% oxygen) conditions, or co-transfected with either control or HIF1α expression plasmid construct (gift from Gregg Semenza, Johns Hopkins, MD). Human control VSMCs were incubated in DMEM containing 2% FBS under either normoxic (21% O2) or hypoxic (1% O2) conditions.
at 24 months of age and APPsw±
at 16 months of age were used to silence Srf
. Dutch/Iowa APP11
at 15 months of age and APPsw±
mice at 16 months of age were used to overexpress Myocd
. All procedures with animals have been preformed using an approved institutional protocol according to the NIH guidelines.
In vivo transduction of cerebral pial arteries
This procedure was performed as we reported previously22
Aβ levels after in vivo gene transfer
We determined endogenous Aβ40 and Aβ42 levels in the vessels and focally in brain tissue in control mice by a sandwich ELISA, as reported previously40
The levels of human Aβ40 and Aβ42 in the Dutch/Iowa APP
mice and APPsw±
mice were determined using ELISA kits KHB3481 and KHB3441, respectively (Biosource), as we reported previously40
. In all studies, Aβ levels were determined focally in brain parenchyma.
Aβ immunostaining of brain tissue in Dutch/Iowa APP and APPsw± mice
Cyrostat sections (10 µm) were stained using polyclonal anti-pan Aβ (1:200; Invitrogen, 44–136) and polyclonal anti-mouse CD31 (1:200; BD Pharmingen, 550274). Fluorescence imaging was obtained using fluorescein goat anti-rabbit IgG (1:200; Invitrogen, F-2765) and Alexa Fluor 594 donkey anti-rat IgG (1:200; Invitrogen, A-21209).
Thioflavin S amyloid staining
Sections were incubated in 0.5% thioflavin S in 70% ethanol for 5 min and washed with distilled water.
Western blotting of isolated brain vessels
Vessels were washed with PBS and lysed with a buffer as above. MYOCD, SRF, SREBP2, LRP and β-actin were detected using the primary antibodies as described above. Primary antibodies for SM-calponin (1:10,000, hCP, Sigma) and SM α-actin (1:1,000, Sigma, A-2547) were also used. Quantification was performed by densitometry analysis using a AlphaImager 2200 and AlphaEase FC software (Alpha Innotech Corporation).
Immunostaining for SREBP2 and LRP in MYOCD-transduced vessels in control mice
Five days after GFP or MYOCD gene transfer, 10-µm frozen sections of brain were stained for SREBP2 nuclear localization and LRP as described above.
Multiphoton in vivo imaging of CAA
We performed a longitudinal 5-day multiphoton in vivo imaging of vascular amyloid in 16-month-old APPsw± mice transduced with Ad.shSRF or Ad.shGFP. Imaging was perfomed on the day of transduction and 5 days after transduction. One day before imaging, animals received an intraperitoneal injection of 10 mg kg−1 methoxy-X04 (Neuroptix). For imaging, mice were anaesthetized with urethane (750 mg kg−1, i.p.) and chloralose (50 mg kg−1, i.p.). Texas Red conjugated Dextran (70K; 200 mg kg−1) was injected into the tail vein to create a fluorescent angiogram. In vivo images were acquired using a Zeiss 5MP multi-photon microscope coupled to a 900-nm mode locked Ti:sapphire laser (Mai Tai, Spectra Physics). Quantification of residual X-04 fluorescence was analysed using NIH Image J software.
In vivo hypoxia model
We used a normobaric chamber with 10% oxygen on the first day, 9% on the second day, and then 8% of oxygen for up to 2 weeks, as we described previously31
ANOVA was used to determine statistically significant differences. P < 0.05 was considered statistically significant.