Apoe mice

ApoE^−/− mice (male, C57BL/6 background, 5 weeks old) were provided by Shanghai Model Organisms Center, Inc. (Shanghai, China) and kept in microisolator cages with water and food available ad libitum, under specific pathogen-free conditions at the Shanghai Model Organisms Center. ApoE^−/− mice were fed a western diet (21% fat, 0.15% cholesterol; SLAC, Shanghai, China) (Liu et al., 2013 (link)) from 5 weeks of age, and samples were collected at 6, 8, 11, 14, and 18 weeks of age (5 mice per group) to determine the timepoint and duration of pharmacological intervention. Thirty-six 5-week-old ApoE^−/− mice were also fed the western diet, and at 14 weeks of age these mice were randomized into four groups. Treatments were administered to the four groups via intraperitoneal injection for 4 weeks, as follows: 1) control (vehicle, 0.25 mL, 2% DMSO/day), 2) Z-YVAD-FMK (Fu et al., 2019 (link)) (0.25 mL, 200 μg/day), 3) Z-LLSD-FMK (0.25 mL, 200 μg/day), and 4) Z-YVAD-FMK + Z-LLSD-FMK (0.25 mL, 200 μg + 200 μg/day). Mice were killed at 18 weeks of age. All mouse studies were approved by the Animal Ethics Committee of Zhongshan Hospital, Fudan University. All experimental procedures were performed in accordance with the guidelines for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996) (Zhang et al., 2021 (link)).

All animal procedures described in this study were performed using 8- to 16-week-old mice and were approved by the Institutional Animal Care and Use Committee of Soochow University (20140431). For platelet specific deletion of CLEC-2 expression, Clec2^fl/fl; Pf4-Cre mice were created by crossing Clec2^fl/flmice with Pf4-Cre mice (stock number 008535; C57BL/6J and Sv129 background; Jackson Laboratory). For hematopoietic deletion of PDPN expression, PDPN^fl/fl; LysM-Cre mice (Mye Pdpn^-/-) were created by crossing PDPN^fl/fl mice with LysM-Cre mice (stock number 004781; C57BL/6J and Sv129 background; Jackson Laboratory). Selplg^-/- mice were obtained from the Jackson Laboratory (stock number 004201, C57BL/6J background). ApoE^-/- mice (stock number T001782, C57BL/6J background), Ldlr^-/- mice (stock number T001464, C57BL/6J background), C57BL/6J mice (stock number N000013) and EGFP (stock number NM-TG-00005, C57BL/6J background) mice were purchased from Shanghai Model Organisms Center. All mice were housed in a pathogen-free facility at an ambient temperature of 22 °C to 25 °C, humidity and light cycle (12:12 h light: dark), and free access to water and food. All mice were backcrossed on C57BL/6J or Sv129 for > 10 generations.

Animal protocols were approved by the Institutional Animal Care and Use Committee of Shanghai Model Organisms Center, Inc., Shanghai, China (Protocol No. 2020-0045, 31 December 2020). Gsdme^−/− mice and ApoE^−/− mice on the C57BL/6J background were obtained from the Shanghai Model Organisms Center, Inc. (Shanghai, China) and crossed to generate Gsdme^−/−/ApoE^−/− mice. The genotyping of Gsdme^−/− mice was performed by amplifying a 920-bp fragment for the wild-type (WT) allele and a 495-bp fragment for the conventional knockout allele using the forward primer TTGGGGCGGGAAAGGTC and the reverse primer AAGCAGGGCAGTTACAGGAG. Only male mice were included. They were fed a WD (21% fat, 0.15% cholesterol, SLAC, Shanghai, China) from 5 to 18 weeks of age and received intraperitoneal injections of SR-717 (0.3% dimethyl sulfoxide, 10 mg/kg per day; MCE, Monmouth Junction, NJ, USA) or phosphate buffer saline (PBS) in the last 4 weeks. ApoE^−/− mice were sacrificed at 10, 14, or 18 weeks, and all Gsdme^−/−/ApoE^−/− mice were sacrificed at 18 weeks.