Rat aneurysm model
Seven-week-old male Sprague−Dawley rats were purchased from Japan SLC (Shizuoka, Japan). Rats were maintained on a light/dark cycle of 12 h/12 h and had free access to chow and water. Under general anesthesia by the inhalation of isoflurane (induction; 5.0 %, maintenance; 1.5~2.0 %), the left common carotid artery (CCA) was cut in the proximal portion and moved to the right side29,30. The left CCA was then anastomosed to the right CCA in an end-to-side fashion with a 10-0 nylon suture to create a bifurcation. Hyper-volemic state was also induced by salt overloading and ligation of the left renal artery. Immediately after the above surgical manipulations, animals were fed the chow containing 8 % sodium chloride and 0.12 % 3-aminopropionitrile (Tokyo Chemical Industry, Tokyo, Japan), an inhibitor of lysyl oxidase that catalyzes the cross-linking of collagen and elastin, which facilitates degenerative changes of arterial walls.
Tissue transparency and immunohistochemistry
Tissue transparency was done using paraformaldehyde-fixed specimens and CUBIC-L and CUBIC-R solutions (#T3740 or #T3741, TCI chemicals, Tokyo, Japan) as manufacture’s instructions. The images were acquired by a confocal laser microscopy (FV3000, Olympus, Tokyo, Japan).
Isolation of cells present in the adventitia of aneurysm walls and preparation of libraries for single cell RNA-sequencing analysis
DiI-containing nanoparticles (Fluorescent (DiI) Control Liposomes (Neutral), #01262101, FormuMax Scientific Inc., Sunnyvale, CA) were intravenously injected in rats to label macrophages infiltrating in lesions. After 3 days, DiI-labelled macrophages were then isolated in a single-cell fashion by the laser-microdissection technique (MMI CellCut Plus, Digital Biology, Tokyo, Japan). The library for single cell RNA-sequencing analysis was prepared by an SMART-Seq Single Cell Plus Kit (#R400751, Takara Bio Inc., Shiga, Japan).
Single cell RNA-sequencing analysis
Using the libraries prepared above, paired-end sequencing (2 × 75 base pair) was performed on a NextSeq500 (Illumina). Each read was then mapped to the Rattus norvegicus reference genome (Rnor6) using CLC genomics workbench (version 11, QIAGEN, Venlo, Netherlands). Differential expression analyses, including principal component analysis and clustering analysis, were performed using the iDEP.951 (http://bioinformatics.sdstate.edu/idep95/). In the analyses, gene expression profile data deposited in the data base was also used (#GSE161798 from Gene Expression Omnibus in National Center for Biotechnology Information, https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE161798).
All the raw data from RNA sequencing analysis was deposited to Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/) (ID # Data will be deposited after the acceptance).
HEK293 cell line and RAW264.7 cell line were maintained in Dulbecco's Modified Eagle's Medium (DMEM) (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) supplemented with 10 % fetal bovine serum (biosera, Nuaille, France).
Cells were treated with DiI-containing nanoparticles (Fluorescent (DiI) Control Liposomes (Neutral), #01262101, FormuMax Scientific Inc.) by adding the particles in the medium for 1 h.
For histological analyses, animals were transcardially perfused with 4 % paraformaldehyde solution after sacrificed by the intraperitoneal administration of a lethal dose of pentobarbital sodium (200 mg/kg). The anterior cerebral artery – olfactory artery bifurcation was then harvested, and 5-µm-thick frozen sections were prepared. In the experiments using culture cells, cells were cultured on a chamber slide (#354631, Corning, Corning, NY) and fixed by 4 % paraformaldehyde. After blocking with 3 % donkey serum (#AB_2337258, Jackson ImmunoResearch, West Grove, PA), slices were incubated with primary antibodies followed by incubation with secondary antibodies conjugated with a fluorescence dye (Jackson ImmunoResearch). In some experiments, the primary antibody conjugated with a fluorescence dye was used. Finally, fluorescent images were acquired on a confocal fluorescence microscope system (FV3000, Olympus, Tokyo, Japan).
The antibodies used were as follows; mouse monoclonal anti-smooth muscle a-actin antibody conjugated with Cy3 (#C6198, Sigma, St. Louis, MI), mouse monoclonal anti-CD68 antibody conjugated with Alexa Fluor 647 (#sc-20060AF647, Santa Cruz Biotechnology, Dallas, TX), rabbit polyclonal anti-S100 antibody (#ab66041, abcam, Cambridge, UK), mouse monoclonal anti-SOX10 antibody (#14-5923-80, Invitrogen, Carlsbad, CA) Alexa Fluor 488-conjugated donkey anti-mouse IgG H&L antibody (#A21202, Thermo Fisher Scientific, Waltham, MA), Alexa Fluor 647-conjugated donkey anti-rabbit IgG H&L antibody (#A31573, Thermo Fisher Scientific).
Scanning Electron Microscopy
The specimens were fixed with 2 % paraformaldehyde and 2 % glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.4) and then stained with 1 % osmium tetroxide (Wako) in 0.1 M sodium phosphate buffer (pH 7.4). After the dehydration in a series of graded ethanol and the replacement by propylene oxide (Nacalai Tesque, Kyoto, Japan), the tissue was coated with a thin layer of platinum palladium using an ion sputter-coater (IB3, Eiko Corporation, Tokyo, Japan). Finally, images were obtained with a high-resolution scanning electron microscopy (S-4700, Hitachi High-Tech Corporation, Tokyo, Japan).