DOI: 10.6084/m9.figshare.16671115.v2
title: In vivo N2 athero data.xlsx

description: Mouse Models. Studies were approved by the Institutional Animal Care and Use Committee of Maine Medical Center. All mice were on the C57BL/6 background. Athero-susceptible mice with inducible SMC-specific Notch2 deletion were generated by crossing Notch2flox/flox×SMMHC-CreERT2 mice (Jackson Laboratory, #01052, #019079) with ApoE-/- mice (Jackson Laboratory #002052). In Notch2flox/flox mice, loxP sites flank exon 3 of Notch2. ApoE-/-×Notch2flox/flox×SMMHC-CreERT2Cre/0 mice were given 1mg tamoxifen i.p. in 100μL corn oil or vehicle control daily for 5d at 5–7 weeks of age. The ApoE-/-×Notch2flox/flox×SMMHC-CreERT2Cre/0 mice are referred to as ApoE-/-;N2SMC-null, and corn-oil treated ApoE-/-×Notch2flox/flox×SMMHC-CreERT2Cre/0 littermates are referred to as ApoE-/-;N2SMC-ctr. Notch2flox/+×SMMHC-CreERT2 mice were treated with 100µL corn oil i.p. daily for 5d at 5–7 weeks of age, followed by retro-orbital injection of PCSK9-AAV 7d later (Boston Children’s Hospital viral core). Dosage was 5 x 1010 gc/mouse injected retro-orbitally. <br><br>Genotyping. Genomic DNA was isolated by alkaline lysis. PCR was performed using 5PRIME MasterMix for the ApoE allele at a 68°C annealing temperature using the primers (5’-GCCTAGCCGAGGGAGAGCCG-3’), (5’-TGTGACTTGGGAGCTCTGCAGC-3’), and (5’-GCCGCCCCGACTGCATCT-3’). This resulted in a 155 bp band from the wildtype and a 245bp product from the mutant allele. PCR to amplify the Notch2 allele was performed at an annealing temperature of 60°C. Notch2fl/fl primers (5’-TAGGAAGCAGCTCAGCTCACAG-3’, 5’-ATAACGCTAAACGTGCACTGGAG-3’) yielded a 161bp band from the wildtype and a 20bp product from the floxed allele. The SMMHC-CreERT2 genotyping was performed at an annealing temperature of 58°C using primers (5’-TCCAACCTGCTGACTGTG-3’, 5’-TCAGAGTTCTCCATCAGGG-3’), yielding a 455bp transgenic product. <br><br>Induction of atherosclerosis and tissue collection. ApoE-/-;N2SMC-ctr and ApoE-/-;N2SMC-null littermates were fed western diet (Research Diets D12079B, 41% calories from fat) for 6 weeks, or fed western diet (Envigo Teklad TD.88137, 42% calories from fat) for 14 weeks. PSCK9-AAV treated mice were fed western diet (Research Diets D12079B, 41% calories from fat) for 6 or 14 weeks. Feeding started 7d after the last injection of tamoxifen/vehicle. Mice were fasted for 12h prior to blood collection to measure fasting glucose (ZOETIS AlphaTRAK® 2) and serum cholesterol (Molecular Probes Amplex Red Cholesterol Assay). Blood was collected by submandibular vein puncture with a sterile lancet (Braintree Scientific) followed by cervical dislocation and perfusion with 10% formalin. The brachiocephalic artery was fixed overnight in 10% formalin and processed for paraffin embedding. The heart was weighed, fixed overnight in 10% formalin and stored in 30% sucrose in PBS. Hearts were bisected horizontally at the atria and the upper portion frozen in OCT (Tissue-Tek) prior to cryosectioning. The spleen was weighed, and left tibia length measured.<br><br>Blood pressure. Systolic and diastolic blood pressure were measured for 5d prior to start of diet and 5d prior to completion of treatment regime using an automated computer controlled Hatteras SC1000 (Hatteras Instruments) analysis system. The first 3d of analysis were training and data were collected the last 2d. Each session consisted of 4 preliminary reads and 10 measurements. <br><br>Analysis of genomic recombination. Two ApoE-/-;N2SMC-null and two ApoE/;N2SMC-ctr mice were collected one week after tamoxifen or corn oil injection. The brachiocephalic artery, brain, and aorta were collected. Brachiocephalic arteries and brain were fixed in 10% formalin before paraffin embedding. The aortic media was isolated by removing endothelium with a cotton swab and dissecting away the adventitia. Aorta and brain were flash frozen. Genomic DNA was isolated by EDTA lysis of 10mg of tissue for 3h or overnight at 55°C, precipitated with 3M sodium acetate, washed with 70% ethanol. PCR of the intact Notch2 floxed allele with genotyping primers results in amplification of a 201bp product, while PCR of excised Notch2 floxed does not, such that after normalizing to an internal control, the relative amplification between samples indicates excision efficiency. Intercellular adhesion molecule 1 (ICAM1) primers (5’-GAAATCATGTCTTGTGGAACTGA-3’, 5’-CTCCTTCAACAGAGAAGCCAG-3’) is a control reaction with matching efficiency. Quantitative PCR was performed using Green Fast qPCR Mix LoRox (Azura) using a CFX384 thermocycler (Bio-Rad). Cycling was performed with an annealing temperature of 60°C for 40 cycles. Notch2 CT values were normalized to ICAM1 CT values and relative presence of intact Notch2 calculated by the comparative CT method (2^−ΔΔCT). <br><br>Tissue lysates and immunoblot. Tissues were ground in liquid nitrogen using RIPA buffer plus protease inhibitors (Sigma), sonicated, and protein quantified using the DC protein assay (Bio-Rad), mixed with Laemmli sample buffer containing 100mM dithiothreitol, and heated at 95°C for 10 minutes. SDS-PAGE was performed using TGX FastCast acrylamide 10% and 12% gels (Bio-Rad) and 15-50µg protein/lane. Gels were transferred to polyvinylidene difluoride membranes using the TransBlot Turbo Transfer System (Bio-Rad). Membranes were blocked for 10 minutes in 5% milk/PBS with 0.01% Tween-20. Primary antibodies were diluted in 5% milk and incubated overnight at 4°C: 0.2μg/mL CST 5732 rabbit anti-Notch2, 0.3μg/mL CST 3700 rabbit anti-β-actin, and 0.5μg/mL Abcam Ab53219 mouse anti-SMMHC. Membranes were incubated for 1h with HRP-linked mouse or rabbit secondary antibodies (CST) diluted in 5% milk. Signal was detected with Luminata Chemiluminescent HRP substrate (Millipore) and imaged on a ChemiDoc MP system (Bio-Rad). <br><br>Histology and immunofluorescence staining. Brachiocephalic arteries were sectioned at 5μm from the aortic arch for ~100μm and stained with H&E. Hearts were cryosectioned through the aortic root, post fixed with 10% formalin fume, and stained with oil-red-O (ORO). Slides were washed in 85% propylene glycol then stained with 0.7% ORO (Sigma) in propylene glycol for 15 min at 55°C. Slides were washed in 85% propylene glycol then distilled water, counterstained with hematoxylin and coverslipped with Aquatex® aqueous mounting media (Millipore). <br>Formalin fixed, paraffin embedded sections were rehydrated and underwent sodium citrate antigen retrieval, 45 minutes permeabilization with 0.5% Triton X-100 (EM Scientific), blocking in PBS with 0.5% Tween-20, 2% BSA (Sigma), and 5% goat serum (Jackson ImmunoResearch) for 2h at room temperature, and with Mouse-on-Mouse Block (Vector Labs) overnight at 4°C. Sections were incubated overnight with primary antibodies in PBS/2% BSA: 1.8μg/mL rabbit anti-Notch2 (CST 4530), 1.0μg/mL rabbit anti-Notch3 (Abcam ab23426), 10μg/mL mouse anti-SMA (Abcam ab7817), rabbit anti-IgG (CST 3900), and 10μg/mL mouse anti-IgG1 (CST 5415), 10μg/mL mouse anti-smooth muscle actin (Abcam ab7817), 2μg/mL rat anti-Mac2 (Cedarlane CL8942AP), 10μg/mL mouse anti-IgG1 (CST 5415), and 2μg/mL rat anti-IgG2a (BioLegend 400502). Sections were washed 3× for 15 minutes in TBS-T and incubated with Alexa Fluor-conjugated secondary antibodies (Invitrogen) in 1% BSA/TBS-T for 1h at room temperature: 2μg/mL goat anti-rabbit-AF488 (A11034) and 2μg/mL goat anti-mouse-AF568 (A11077), 2μg/mL goat anti-mouse-AF488 (A11001), and 2μg/mL goat anti-rat-AF568 (A11004). <br>Aortic root sections were permeabilized for 5 minutes with 0.1% Tween20, then treated with 0.01mol/L sodium citrate buffer at 50°C 15 minutes. Sections were blocked in 1% BSA/TBS-T for 20 minutes before overnight incubation at 4°C with primary antibodies in 1% BSA/TBS-T: 0.12μg/mL rabbit anti-smooth muscle actin (CST 19245), 2μg/mL rat anti-Mac2 (Cedarlane CL8942AP), 0.12μg/mL rabbit anti-IgG (CST 3900), and 2μg/mL rat anti-IgG2a (BioLegend 400502). Secondary antibodies (Invitrogen) were used in 1% BSA/TBS-T for 1h at room temperature: 2μg/mL goat anti-rabbit-AF488 (A11034) and 2μg/mL goat anti-rat-AF568 (A11004). Sections were washed and incubated for 2 minutes with TrueVIEW Autofluorescence Quenching Kit and coverslipped using Vectashield Hard Set anti-fade mounting medium (Vector Labs). Confocal images were captured using a Leica TCS SP8 laser scanning confocal microscope with a 10×/0.40 dry or a 63x/1.40 oil objective. <br><br>Morphometry and image analysis. Quantification was performed while blinded to group. All brachiocephalic artery sections were quantified, while for aortic roots, sections 100μm-300μm from appearance of first leaflet were used. Images were analyzed for plaque area, medial area, and necrotic core, and ORO images were also analyzed for lipid content. Images were acquired as 18μm z-stacks composed of 7 slices interspaced by 3μm, then sum slices was applied to generate 32-bit greyscale sum projections in ImageJ. Images were also analyzed for smooth muscle actin and Mac2 staining. Plaque and medial areas were normalized to vessel circumference. Morphometry was performed by tracing plaques, necrotic cores, and the internal and external elastic lamina. The ROI (region of interest) manager was used. For aortic roots, medial area was traced to include only areas of the vessel wall with dense elastic fiber. For ORO images, color thresholding within red hued pixels allowed for selection of ORO positive area. The smooth muscle actin and Mac2 sum projections were thresholded using the Otsu algorithm.