Supplemental data - Circulation：补充数据循环

Supplemental data - Circulation：补充数据循环 Supplemental Data Methods Animal protocol -/-We studied dynamic osteogenic changes in carotid arteries of apoE mice (20- 30, n=35 and 72 weeks of age, n=5) that consumed an atherogenic diet (Teklad TD 88137; 42% milk fat, 0.2% total cholesterol, Harlan, Indianapolis, IN) from 10 weeks of age. Mice were randomized either to continue with the atherogenic high cholesterol diet (n=15, sequential imaging group; n=5, cathepsin imaging group; n=5, histological control group) or to consume the high-cholesterol diet admixed with an HMG-CoA reductase inhibitor (n=10, statin group, 0.01% w/w atorvastatin, Pfizer, Groton, CT). Age-matched -/-wild-type C57/BL6 mice (n=5, Jackson Laboratory, Bar Harbor, ME) and apoE mice lacking probe injection (n=5) served as controls. The Subcommittee on Research Animal Care at Massachusetts General Hospital approved all procedures. Intravital laser scanning fluorescence imaging and three-dimension reconstruction Mice received imaging agents or saline via intravenous injection 24 hours before imaging. After surgically isolating the right carotid artery under anesthesia (isofluorane, 2% vol supplemented with oxygen), we performed multichannel fluorescence imaging using an intravital laser scanning fluorescence microscope (IV100, Olympus Corporation, Tokyo, Japan) specifically developed for imaging small experimental animals. We collected images through the vessel at 3.2 ,m intervals along the z-axis using a 4x UplanApo (N.A. 0.16) objective. Excitation at 488 nm, 633 nm and 748 nm and image collection of the different channels was done serially to avoid crosstalk between channels. We used dichroic mirror SDM570 and barrier filter BA650-700 nm for the fluorescent noanoparticle 680 signal and dichroic mirror SDM750 and BA770 long pass filter for the OsteoSense750 signal. In plane resolution was 12.5 µm. Image stacks were processed and analyzed using ImageJ software (v.1.38a, Bethesda, MD). For 3D reconstruction image stacks were split into individual channels and imported into AMIRA (v. 3.1, Mercury Computer Systems, France). All images were leveled identically. We registered colocalization when two fluorescence emission signals (green, inflammation; red, calcification) overlapped within the same 3D image using 1ImageJ, colocalization plugin . Two grey-scale images were merged to a red-green image and colocalized pixels were highlighted in white. To superimpose 20 and 30-week 3D reconstructions, we adjusted vessel positions using ImageJ software until vessels showed good overlap, and then transferred them to AMIRA for reconstruction. In vivo computed tomography imaging Mice anesthetized with isoflurane underwent noninvasive micro-CT imaging (Gamma Medica, Northridge, CA) of vascular calcification followed by intravital laser scanning fluorescence microscopy. We collected 256 projections with delay time between projections of 0.02 sec. We used a source voltage of 50.0kVp with a current of 500uA. The image resolution was 72 ,m isotropic. To visualize vascular anatomy, we performed imaging immediately after injection of 250 ,L iodenated CT contrast agent. 3D CT datasets were displayed using OsiriX shareware (v 2.6, OsiriX Medical Imaging Software). Macroscopic fluorescence reflectance imaging ex vivo After mice were euthanized, aortas were perfused with saline, dissected and imaged to map the macroscopic NIRF signals elaborated from each imaging agent using a fluorescence reflectance imaging system equipped with multichannel filter sets, including green (GFP/FITC; excitation, 406 to 450 nm; emission, 495 to 525 nm), far red (VT680; excitation, 615 to 645 nm; emission, 680 to 720 nm), and near-infrared (ICG; excitation, 716 to 756 nm; emission 780 to 820 nm) (Omega Optical, Brattleboro, VT). Fluorescence images were obtained with an exposure time of 60 seconds to 4 minutes. Subsequently, aortas with carotid arteries were dissected and processed for histological analysis. Flow cytometry Aortas were excised and placed into a cocktail of collagenase I, collagenase XI, DNase I and hyaluronidase (Sigma-Aldrich, Inc.) at 37:C for 1 h, as described elsewhere 2. Cells were then triturated through nylon mesh (BD Biosciences, San Jose, CA). Spleens were removed, triturated in HBSS (Cellgro Mediatech, Inc., Herndon, VA) at 4:C with the end of a 3 ml syringe, and filtered through nylon mesh. The cell suspensions were centrifuged (15 min, 500 ， g, 4:C), and the resulting single cell suspensions washed with HBSS supplemented with 0.2% (w/v) BSA and 1% (w/v) FCS. Cells then were incubated with monoclonal antibodies against myeloid cells (CD11b-APC, M1/70) (BD Biosciences), macrophages/dendritic cells (F4/80 (BM8)-biotin-Strep- bPerCP, I-A (AF6-120.1)-biotin-Strep-PerCP and CD11c (HL3)-biotin-Strep-PerCP) (BD Biosciences) or osteoblast-like cells (osteopontin (BDN04)-biotin-Strep-PerCP) (R&D Systems, Minneapolis, MN). Intracellular staining of osteopontin was performed with a Cyotifx/Cytoperm Kit (BD Biosciences). OsteoSense was detected using 695/40 and 685LP filter configuration. Data were acquired on an LSRII (BD Biosciences) and analyzed by FlowJo software (v8.1.1, Treestar) Correlative histopathological assessment i) Morphological characterization. Tissue samples were frozen in OCT compound (Sakura Finetech, Torrance, CA) and 5 µm serial sections were cut and stained with hematoxylin and eosin for general morphology. Alkaline phosphatase activity (early marker of osteoblastic differentiation) was detected on cryosections 3according to manufacturer instructions (Vector Labs, Burlingame, CA) . Von Kossa silver stain histochemically imaged inorganic phosphate and Alizarin Red detected 3calcium deposition on adjacent sections. ii) Multichannel fluorescence microscopy. Unstained sections were imaged using an upright epifluorescence microscope (Eclipse 80i, Nikon Instruments, Melville, New York) with a cooled CCD camera (Cascade, Photometrics, AZ). Fluorescence images were obtained at a wavelength of green (filter, 480?20 nm excitation; 535?25 nm emission, Q505LP bandpass), far red (filter, 650?22.5 nm excitation; 710?25 nm emission, Q680LP bandpass) or near-infrared (filter, 775?25 nm excitation; 845?27.5 nm emission, Q810LP bandpass) depending on the probe. We used the same exposure time (1000 msec) for each imaging agent. iii) Immunohistochemistry. Validation of NIRF signals employed immunohistochemistry for macrophages (anti-mouse Mac3, BD Biosciences, San Jose, CA; anti-human CD68, Dako, Carpinteria, CA), cathepsin K (Santa Cruz Biotechnology, Santa Cruz, CA), osteoblast differentiation markers (osteopontin and osteocalcin, Abcam, Cambridge, MA) and osteogenic transcription factor (Cbfa1/Runx2, RD Systems, Minneapolis, MN). Immunohistochemistry used avidin-biotin peroxidase method. The reaction was visualized with a 3-amino-9-athyl-carbazol substrate (AEC, Sigma Chemical, Saint Louis, Missouri). Adjacent sections treated with nonimmune IgG provided controls for antibody specificity. Images were captured with a digital camera (Nikon DXM 1200-F, Nikon Inc, Melville, NY). iv) Transmission electron microscopy. Tissue processed for electron microscopy was fixed with 2% glutaraldehyde in 0.1 M cacodylate buffer followed by postfixation in 2.0% osmium tetroxide. Tissue was dehydrated in ethanol, treated in propylene oxide, and embedded in Poly/Bed 812 (Polysciences Inc., Warrington, Pennsylvania). All specimens were rinsed and stained en bloc with uranil acetate before ethanolic dehydration. Thin sections were cut at 60 nm, stained with lead citrate and uranyl acetate, and examined with a JOEL-1000CX transmission microscope (JOEL USA, Inc., Peabody, MA). Molecular imaging agents i) Macrophage-targeted fluorescent nanoparticles (FNP). We used cross- linked iron oxide fluorescent nanoparticle, an agent that elaborates fluorescence detectable through the NIRF window (ex/em 673/694 nm) for in vivo detection of 3, 4macrophage accumulation . Mice received NIRF nanoparticle (15 mg/kg iron) via tail vein injection 24 hours before imaging. ii) Calcification. We used bisphosphonate-conjugated imaging agent (OsteoSense750, VisEn Medical Inc., Woburn, MA), which elaborates fluorescence 3, detectable through the NIRF window (ex/em 750/780 nm) to detect osteoblastic activity 5, 6. Mice received OsteoSense750 (2 nmol/150 mL) via tail vein injection 24 hours before imaging. iii) Cathepsin K activity. Protease-activatable imaging agent detects the 7activity of cathepsin K in atherosclerotic lesions . Cathepsin K probe consists of backbone of a cathepsin K-cleavable peptide substrate containing a fluorochrome. Following enzymatic cleavage, the fluorochromes separate, resulting in amplification of the signal. This agent elaborates fluorescence detectable through the NIRF window (ex/em 673/694 nm). Mice received cathepsin K agent (5 nmol/150 mL) via tail vein injection 24 hours prior imaging. Imaging inflammation and calcification in human atherosclerotic specimens ex vivo Human carotid endarterectomy specimens (n=6) were dissected and incubated in oDMEM at pH7.4 (n=3) or in a mixture of imaging agents (n=3) in 5% CO at 37C. 2 Fluorescence reflectance imaging was acquired at 2, 12, and 24 hours. Specimens were then frozen in OCT compound and sectioned for histological analysis. The specimens were obtained according to the Institutional Review Board protocol approved by the Human Research Committee at Massachusetts General Hospital. Cell culture Human mononuclear cells were isolated from buffy coats (Research Blood Components, Brighton, MA) by density gradient centrifugation and adherence, and 8cultured in RPMI-1640 containing 5% human serum and 1% penicillin/streptomycin . At day 14, differentiated macrophages underwent serum starvation with no human serum for 24 hrs. After replacement of the serum starvation media by the fresh RPMI with no serum and another 24 hr incubation, macrophage-conditioned media were collected. Cell-free media were used as control. Human primary aortic smooth muscle cells were obtained from discarded surgical specimens in accordance with a protocol approved by the IRB of the Brigham and Women’s Hospital. Smooth muscle cells at passage 2-3 were cultured with macrophage-conditioned media or control media for 6 hrs. Real-time RT-PCR Total RNA was isolated from smooth muscle cells using QIAshredder and RNeasy Mini kit (Qiagen, Valencia, CA). Oligo(dT) primer, dNTP, and SuperScript II 12-18 reverse transcriptase (Invitrogen, Carlsbad, California) were used to generate cDNA. PCR employed SYBR Green Master Mix and MyiQ Single-Color Real-Time PCR Detection System (BioRad, Hercules, CA). Oligonucleotide primers for detection of human alkaline phosphatase cDNA were: forward, 5’-AGACTGCGCCTGGTAGTTGT-3’ and reverse, 5’-CCACGTCTTCACATTTGGTG-3’. Quantitative PCR threshold cycles (C) were GAPDH-normalized (,,C) and relative fold changes calculated by TT -,,CTcomparative C method, 2. T Quantitative assessment and statistical analyses. Macrophage, von Kossa, ALP and OsteoSense positive plague areas were calculated as % positive area to total plaque area using IPLab imaging software (version 3.9.9; Scanalytic, Inc., Rockville, MD). For imaging, we defined plaque regions by the fluorescent nanoparticle- or OsteoSense-derived signals selected in each sum-image using imaging software ImageJ (v. 1.38a, Bethesda, MD). The total pixel intensity of all pixels in the selected area (signal intensity) was used for comparisons between samples. The target-to-background ratio was calculated as (plaque signal)/(adjacent vessel background signal) using ImageJ software. Colocalization was analyzed using ImageJ 1colocalization plugin using Pearson’s and overlap coefficients . Pearson’s coefficient indicates the linear correlation coefficient between pixel intensities in the red and green channels (-1 corresponds to no colocalization, 1- to perfect colocalization). Overlap coefficient is similar to Pearson’s coefficient, except the mean values are not subtracted (0 corresponds to no colocalization, 1 – to perfect colocalization). Statistical analyses for comparison of multiple groups used one-way ANOVA followed by the Tukey post-hoc test performed with GraphPad Prism (v. 4.0, GraphPad Software, San Diego, CA). ,,C T of real-time RT-PCR data for alkaline phosphatase mRNA were used in Mann-Whitney’s U-test. Data are presented as mean ?SEM. P-values less than 0.05 were considered significant. References 1. Bolte S, Cordelieres FP. A guided tour into subcellular colocalization analysis in light microscopy. J Microsc. 2006;224:213-232. 2. 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