Smooth muscle-derived macrophage-like cells contribute to multiple cell lineages in the atherosclerotic plaque

Smooth Muscle-derived Macrophage-like Cells Contribute to Multiple Cell Lineages in the Atherosclerotic Plaque Yi Li1,2, Huan Zhu2, Qianyu Zhang3, Ximeng Han2, Zhenqian Zhang2, Linghong Shen1, Lixin Wang4, Kathy O. Lui5, Ben He1, Bin Zhou2,3,6 1Department of Cardiology, Shanghai Chest Hospital, Shanghai Jiaotong University, Shanghai, China. 2State Key Laboratory of Cell Biology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences; University of Chinese Academy of Sciences, Shanghai 200031, China. 3School of Life Science and Technology, ShanghaiTech University, 100 Haike Road, Shanghai 201210, China. 4Department of Cardiac Surgery, Zhongshan Hospital, Fudan University, Shanghai, China. 5Department of Chemical Pathology; and Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, Hong Kong SAR, 999077, China 6School of Life Science, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Hangzhou 310024, China.


Methods and Materials
The data, analytic methods, and study materials that support the findings of this study are available from the corresponding author upon reasonable request.

Experimental mice
All mouse experiments were done with the approval of the Institutional Animal Care and Use Committee (IACUC) at the Institutes for Biochemistry and Cell Biology, Shanghai institutes for Biological Sciences, Chinese Academy of Sciences. Both male and female mice were analyzed in this study. The mouse background is a mixture of C57BL/6J and ICR. The Myh11-Dre mouse line was generated by knock-in of Dre cDNA through homologous recombination using CRISPR/Cas9 strategy and direct editing in mouse zygotes. We inserted Dre-WPRE-polyA sequence into the Myh11 gene locus by replacing ATG of endogenous gene. 4 Myh11-Dre knock-in mice were got from 21 zygotes. The plasmid containing Dre was originally obtained from Konstantinos Anastassiadis and Francis Stewart at Dresden, Germany. The CD11b-CrexER mouse line was generated by knock in of Cre-rox-ER-rox cDNA through homologous recombination using CRISPR/Cas9 strategy and direct editing in mouse zygotes. After characterizing, we got 1 CD11b-CrexER knock-in mice from 122 zygotes. We generated Cre-rox-ER-rox-WPRE-polyA cassette and insert it into CD11b gene locus by replacing the endogenous ATG. Shanghai Model Organisms Center, Inc. (SMOC) generated the Myh11-Dre and CD11b-CrexER mouse line. LDLR-/-mice were reported previously 1 . The mice were fed on high fat diets (Dyets, ASHF3, 1.25% cholesterol) for 16-32 weeks for induction of atherosclerosis and normal laboratory diet (Jiangsu Xietong Pharmaceutical Bioengineering, 1010085) as control.

Immunofluorescent staining
Tissues were collected in PBS, and then fixed in 4% PFA for 50-60 minutes in 4℃. After three times of washing in PBS for 15 minutes, tissues were dehydrated in 30% sucrose (dissolved in PBS) for 6-8 hours and embedded in OCT (Sakura). Next, cryosections of 9-10μm in thickness were collected. After air dry for 1-1.5 hour at room temperature, sections were incubated with 30% H2O2 for 15 minutes. After two times of washing in PBS for 15 min at room temperature, slides were blocked in blocking buffer (5% donkey serum, 0.1% Triton X-100 in PBS) for 40 min at room temperature, followed by incubation with primary

Aortic cell isolation and flow cytometry
The aortas were harvested from the mice perfused with PBS, and then finely minced and transferred to the digestion mix (HBSS, 2U/ml Liberase (5401127001, Sigma) and 2 U/ml elastase (LS002279, Worthington)). Tissues were incubated with the digestion mix at 37℃ for 30-40 minutes. The cell suspension was filtered through 70 µm cell strainer and then pelleted by centrifugation at 400g for 5min.
The precipitated cells were re-suspended with PBS and then stained with violet dye at 4℃ for 30 minutes.

Statistical analysis
All data were representative of 5 individuals, as indicated in each figure legend, and presented as mean values ± SEM.