In vivo N2 athero data.xlsx

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&amp;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.

小鼠模型<br><br>本研究经缅因医学中心实验动物护理与使用委员会审批通过。所有实验小鼠均为C57BL/6背景品系。通过将Notch2flox/flox×SMMHC-CreERT2小鼠（购自杰克逊实验室，货号#01052、#019079）与ApoE基因敲除（ApoE-/-）小鼠（购自杰克逊实验室，货号#002052）杂交，构建可诱导平滑肌细胞（Smooth Muscle Cell, SMC）特异性Notch2缺失的动脉粥样硬化易感小鼠模型。在Notch2flox/flox小鼠中，loxP位点序列位于Notch2基因第3外显子的两侧。<br><br>对5~7周龄的ApoE-/-×Notch2flox/flox×SMMHC-CreERT2Cre/0小鼠，每日腹腔注射（intraperitoneal, i.p.）1mg他莫昔芬（溶于100μL玉米油中）或溶剂对照，连续给药5天。该ApoE-/-×Notch2flox/flox×SMMHC-CreERT2Cre/0小鼠被命名为ApoE-/-;N2SMC-null，而经玉米油处理的同窝ApoE-/-×Notch2flox/flox×SMMHC-CreERT2Cre/0小鼠则作为对照，命名为ApoE-/-;N2SMC-ctr。<br><br>对5~7周龄的Notch2flox/+×SMMHC-CreERT2小鼠，每日腹腔注射100μL玉米油，连续给药5天；7天后经眶后静脉注射PCSK9-AAV（购自波士顿儿童医院病毒核心实验室），注射剂量为每只小鼠5×10^10基因组拷贝（gc）。<br><br>基因分型<br><br>采用碱裂解法提取基因组DNA。针对ApoE等位基因的PCR反应采用5PRIME MasterMix预混体系，退火温度设为68℃，所用引物为(5’-GCCTAGCCGAGGGAGAGCCG-3’)、(5’-TGTGACTTGGGAGCTCTGCAGC-3’)及(5’-GCCGCCCCGACTGCATCT-3’)。野生型等位基因可扩增出155bp的条带，突变型等位基因则扩增出245bp的产物。<br><br>针对Notch2等位基因的PCR扩增退火温度设为60℃。使用Notch2fl/fl特异性引物(5’-TAGGAAGCAGCTCAGCTCACAG-3’、5’-ATAACGCTAAACGTGCACTGGAG-3’)时，野生型等位基因扩增出161bp条带，flox型等位基因则扩增出20bp产物。针对SMMHC-CreERT2的基因分型反应退火温度设为58℃，所用引物为(5’-TCCAACCTGCTGACTGTG-3’、5’-TCAGAGTTCTCCATCAGGG-3’)，可扩增出455bp的转基因特异性条带。<br><br>动脉粥样硬化造模与组织采集<br><br>ApoE-/-;N2SMC-ctr与ApoE-/-;N2SMC-null同窝小鼠分为两组：一组喂食西方高脂饲料（Research Diets公司货号D12079B，脂肪供能比41%）6周，另一组喂食西方高脂饲料（Envigo Teklad公司货号TD.88137，脂肪供能比42%）14周。经PCSK9-AAV处理的小鼠则喂食Research Diets公司货号D12079B的西方高脂饲料（脂肪供能比41%），分别持续6周或14周。高脂饲料喂养于最后一次他莫昔芬/溶剂注射后7天开始。<br><br>采血前小鼠禁食12小时，采集的血液用于检测空腹血糖（使用ZOETIS AlphaTRAK® 2血糖仪）与血清总胆固醇（采用Molecular Probes Amplex Red胆固醇检测试剂盒）。采血采用无菌柳叶刀（Braintree Scientific公司）行颌下静脉穿刺，随后通过颈椎脱臼法处死小鼠，并用10%福尔马林进行心脏灌注固定。头臂干动脉于10%福尔马林溶液中固定过夜，后续用于石蜡包埋处理。称量心脏重量后，于10%福尔马林溶液中固定过夜，随后置于含30%蔗糖的PBS溶液中保存。心脏于心房水平处横向剖切，上部组织经OCT包埋剂（Tissue-Tek）速冻后用于冰冻切片。称量脾脏重量，并测量左侧胫骨长度。<br><br>血压检测<br><br>在高脂饲料喂养开始前5天，以及造模方案完成前5天，采用计算机自动化控制的Hatteras SC1000血压分析系统（Hatteras Instruments公司）检测小鼠收缩压与舒张压。检测前3天为适应性训练阶段，最后2天采集有效数据。每次检测包含4次预读与10次正式测量。<br><br>基因组重组效率分析<br><br>在他莫昔芬或玉米油注射后1周，采集2只ApoE-/-;N2SMC-null小鼠与2只ApoE-/-;N2SMC-ctr小鼠的组织。采集头臂干动脉、脑组织与主动脉组织。头臂干动脉与脑组织于10%福尔马林溶液中固定后进行石蜡包埋。通过棉签擦拭去除内皮细胞，并剥离外膜，以分离主动脉中膜组织。主动脉与脑组织经快速速冻后保存。<br><br>采用EDTA裂解法提取基因组DNA：取10mg组织，于55℃裂解3小时或过夜，随后用3M乙酸钠沉淀DNA，并用70%乙醇洗涤沉淀。使用基因分型引物扩增完整的Notch2 floxed等位基因可得到201bp的产物，而经重组切除的Notch2 floxed等位基因则无法被扩增。因此，以内部参照基因标准化后，不同样本间的相对扩增强度可反映Notch2基因的切除效率。<br><br>细胞间黏附分子1（Intercellular Adhesion Molecule 1, ICAM1）的引物序列为(5’-GAAATCATGTCTTGTGGAACTGA-3’、5’-CTCCTTCAACAGAGAAGCCAG-3’)，作为扩增效率匹配的内参对照。定量PCR（qPCR）采用Green Fast qPCR Mix LoRox预混液（Azura公司），在CFX384实时荧光定量PCR仪（Bio-Rad公司）上进行。PCR循环程序为：退火温度60℃，共40个循环。将Notch2基因的CT值以ICAM1基因的CT值进行标准化，采用相对定量CT法（2^−ΔΔCT）计算完整Notch2等位基因的相对含量。<br><br>组织裂解与免疫印迹实验<br><br>组织经液氮研磨后，采用含蛋白酶抑制剂（Sigma公司）的RIPA裂解液进行裂解，超声处理后采用DC蛋白定量试剂盒（Bio-Rad公司）测定蛋白浓度。将蛋白样品与含100mM二硫苏糖醇的Laemmli上样缓冲液混合，于95℃加热10分钟变性。采用10%与12%的TGX FastCast丙烯酰胺凝胶（Bio-Rad公司）进行SDS-聚丙烯酰胺凝胶电泳（SDS-PAGE），上样量为每泳道15~50μg蛋白。<br><br>采用TransBlot Turbo转印系统（Bio-Rad公司）将凝胶中的蛋白转移至聚偏二氟乙烯（PVDF）膜上。PVDF膜于含0.01% Tween-20的5%脱脂牛奶PBS溶液中封闭10分钟。一抗用5%脱脂牛奶稀释，于4℃孵育过夜：0.2μg/mL CST 5732兔抗Notch2抗体、0.3μg/mL CST 3700兔抗β-肌动蛋白抗体，以及0.5μg/mL Abcam Ab53219小鼠抗SMMHC抗体。随后用5%脱脂牛奶稀释的HRP标记山羊抗兔或抗小鼠二抗（CST公司）室温孵育1小时。采用Luminata化学发光HRP底物（Millipore公司）显影信号，并通过ChemiDoc MP成像系统（Bio-Rad公司）采集图像。<br><br>组织学与免疫荧光染色<br><br>从主动脉弓起始处开始，对头臂干动脉进行连续5μm切片，连续切片长度约100μm，随后进行苏木精-伊红（H&E）染色。心脏经冰冻切片至主动脉根部区域，用10%福尔马林蒸汽复固定后，进行油红O（Oil Red O, ORO）染色。切片经85%丙二醇洗涤后，置于55℃的0.7%油红O丙二醇溶液中染色15分钟。随后用85%丙二醇及蒸馏水洗涤切片，经苏木精复染后，采用Aquatex®水性封固剂（Millipore公司）封片。<br><br>福尔马林固定、石蜡包埋的切片经脱蜡水化后，采用柠檬酸盐缓冲液进行抗原修复；用0.5% Triton X-100（EM Scientific公司）透化处理45分钟，随后于室温下在含0.5% Tween-20、2%牛血清白蛋白（BSA，Sigma公司）及5%山羊血清（Jackson ImmunoResearch公司）的PBS溶液中封闭2小时；再用Mouse-on-Mouse封闭液（Vector Labs公司）于4℃封闭过夜。<br><br>切片于含2% BSA的PBS溶液中，与以下一抗于4℃孵育过夜：1.8μg/mL兔抗Notch2抗体（CST 4530）、1.0μg/mL兔抗Notch3抗体（Abcam ab23426）、10μg/mL小鼠抗平滑肌肌动蛋白（Smooth Muscle Actin, SMA）抗体（Abcam ab7817）、兔抗IgG抗体（CST 3900）、10μg/mL小鼠抗IgG1抗体（CST 5415）、10μg/mL小鼠抗平滑肌肌动蛋白抗体（Abcam ab7817）、2μg/mL大鼠抗Mac2抗体（Cedarlane CL8942AP）、10μg/mL小鼠抗IgG1抗体（CST 5415）以及2μg/mL大鼠抗IgG2a抗体（BioLegend 400502）。切片经TBS-T缓冲液洗涤3次，每次15分钟；随后用1% BSA/TBS-T稀释的Alexa Fluor标记二抗（Invitrogen公司）室温孵育1小时：2μg/mL山羊抗兔AF488抗体（A11034）、2μg/mL山羊抗小鼠AF568抗体（A11077）、2μg/mL山羊抗小鼠AF488抗体（A11001）以及2μg/mL山羊抗大鼠AF568抗体（A11004）。<br><br>主动脉根部切片经0.1% Tween-20透化处理5分钟，随后置于0.01mol/L柠檬酸盐缓冲液中，于50℃孵育15分钟进行抗原修复。切片于1% BSA/TBS-T缓冲液中封闭20分钟，随后与以下一抗于1% BSA/TBS-T缓冲液中4℃孵育过夜：0.12μg/mL兔抗平滑肌肌动蛋白抗体（CST 19245）、2μg/mL大鼠抗Mac2抗体（Cedarlane CL8942AP）、0.12μg/mL兔抗IgG抗体（CST 3900）以及2μg/mL大鼠抗IgG2a抗体（BioLegend 400502）。随后用1% BSA/TBS-T稀释的二抗（Invitrogen公司）室温孵育1小时：2μg/mL山羊抗兔AF488抗体（A11034）与2μg/mL山羊抗大鼠AF568抗体（A11004）。<br><br>切片洗涤后，用TrueVIEW自发荧光淬灭试剂盒孵育2分钟，随后采用Vectashield Hard Set抗荧光淬灭封固剂（Vector Labs公司）封片。共聚焦图像采用Leica TCS SP8激光扫描共聚焦显微镜采集，分别使用10×/0.40干镜物镜或63×/1.40油镜物镜。<br><br>形态计量学与图像分析<br><br>所有定量分析均采用单盲法进行。所有头臂干动脉切片均进行定量分析；对于主动脉根部，则选取从首个瓣膜瓣叶出现位置起100μm~300μm范围内的切片进行分析。图像分析指标包括斑块面积、中膜面积、坏死核心面积，油红O染色图像额外分析脂质沉积面积。<br><br>图像以18μm的Z轴堆叠序列采集，包含7层间隔3μm的切片；随后在ImageJ软件中采用求和切片（sum slices）算法生成32位灰度叠加投影图像。同时对平滑肌肌动蛋白与Mac2染色的图像进行分析。斑块面积与中膜面积均以血管周长进行标准化校正。形态计量学分析通过手动勾画斑块、坏死核心、内弹性膜与外弹性膜的轮廓完成，使用ImageJ的感兴趣区域（Region of Interest, ROI）管理器工具。<br><br>对于主动脉根部样本，中膜面积仅勾画含有密集弹性纤维的血管壁区域。油红O染色图像通过红色像素阈值分割，选取油红O阳性染色区域。平滑肌肌动蛋白与Mac2染色的叠加投影图像采用Otsu算法进行阈值分割。