Human samples
In this study, total 33 atheromatous coronary arterial specimens were obtained from cardiac transplant patients diagnosed with severe ASCVDs at the Department of Cardiovascular Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology (Wuhan, China), as described previously.63 This study (Approval No. 2021-0189) was conducted under the permission of the Ethics Committee of Union Hospital, Tongji Medical College, Huazhong University of Science and Technology (Wuhan, China). Briefly, recipient hearts were collected during surgery, and immediately immersed in ice-cold sterile saline. The left circumflex, left anterior descending, and right coronaries were excised and sectioned into small segments of 5 mm in length under sterile conditions. Coronary segments were fixed in 4% paraformaldehyde (PFA) or preserved at −80 °C for further detection. All patients and/or their immediate family members were informed of this experiment and signed the informed consent form. Detailed patient demographics and clinical features are tabulated in Table S1.
Bacterial culture
Pg W83 was kindly provided by West China College of Stomatology, Sichuan University, China. The gingipains (Rgp & Kgp)-deficient Pg mutant strain KDP136 was kindly provided by Hospital of Stomatology, Sun Yat-sen University, China. Pg W83 and KDP136 were grown on Columbia blood agar plates (CP0160, Huankai Microbial, China) at 37 °C anaerobically for 5–7 days, and sub-cultured in hemin- and vitamin K (5 g/mL each)-supplemented brain heart infusion (BHI) medium (BD) under 37 °C anaerobic condition for 1 day.
Rabbit model studies
Male New Zealand white rabbits (2.5–3.0 kg) were obtained from Wuhan Wonderjourney Biotechnology Co., Ltd. This experiment was conducted under the permission of the Institutional Animal Care and Use Committee (IACUC) of Tongji Medical College, Huazhong University of Science and Technology (IACUC number 4137).
To address the relationship between Pg and atherosclerotic plaque instability, rabbits were randomly assigned to three groups: untreated, periodontitis, and periodontitis with Pg infected groups (n = 6 in each group). In the periodontitis group, a 3-0 silk suture was ligated around bilateral mandibular second premolars. General anesthesia was administered via isoflurane inhalation (1.5–3%). In periodontitis with Pg-infected group, rabbits received periodontal ligature and additional topical applications of Pg-containing slurry to the ligature sites three times per week. The untreated group underwent no periodontal ligature. All experimental rabbits were individually housed with free water access and fed 0.5% cholesterol-supplemented high-fat diet for 16 weeks.
At the end of the experiment, rabbits were euthanized, and aortic arch specimens were harvested and fixed in 4% PFA for further analyses.
Mouse model studies
6-week-old male C57BL/6 J Apoe−/− mice were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. These studies were carried out under the permission of the IACUC of Tongji Medical College, Huazhong University of Science and Technology (IACUC no. 3611). Because the protective effect of estrogen in ASCVDs in female, only male animals were involved in our experiments. To determine the appropriate group size, we reviewed the literature on atherosclerosis and noticed that six mice each group was acceptable.64,65 And by statistical analysis, we calculated the group sizes by using the formula (n = [(Z1 − α/2 + Z1 − β) (κ − 1)/(δ/σ)],2 α = 0.05 (two-tailed), β = 0.90, n indicates the sample size of each group, κ indicates the number of groups, δ indicates population difference of research significance, σ indicates population standard deviation) with the data of previous literature, and the result showed that five mice each group was statistically enough in our cases.66
Mice were housed under specific pathogen-free condition (23 ± 1 °C, 12 h light/dark cycle) under ad libitum access to both water and a western diet (D12079B, Jiangsu Xietong Pharmaceutical Bio-engineering Co., Ltd, China).
The inclusion criteria were all animals that survived all experimental procedures and completed the full experimental period. Animals failing to survive the study duration were excluded. Mice were subjected to the following experiments:
For analysis of the relationship between Pg infection and atherosclerotic plaque destabilization, Apoe−/− mice were untreated, or intravenously injected with 107 CFU Pg (suspended in 0.1 mL PBS) or PBS once per week for 20 weeks, 14 weeks, or 8 weeks. The untreated group underwent no injection.
To confirm the role of macrophage in Pg-induced atherosclerotic plaque destabilization, Apoe−/− mice were administrated intraperitoneally with LC (0.2 mL, F70101C-A-2, FormuMax) for mononuclear phagocyte depletion, or vehicle once per 3 days, and intravenously injected with 107 CFU Pg suspension or PBS once a week for 8 weeks. The first administration of LC was performed 24 h before Pg infection. The untreated group accepted no injection.
To investigate the effect of oxidative stress on Pg-promoted atherosclerotic plaque destabilization, EUK134 (10 mg/kg, S4261, Selleck) or DMSO (as control) was delivered via intraperitoneal injection to Apoe−/− mice once per week, combined with or without intravenous injection with 107 CFU Pg once per week for 8 weeks. The administration of EUK134 was performed 1 h before Pg infection. The untreated group underwent no injection.
To validate the role of gingipains in Pg-induced atherosclerotic plaque destabilization, Apoe−/− mice were intravenously injected with or without 107 CFU Pg or KDP136 suspensions, or PBS once a week for 8 weeks. The untreated group accepted no injection.
To identify the role of cell lipid uptake in Pg-caused atherosclerotic plaque destabilization, fucoidan (60 mg/kg, E0365, Selleck) or PBS was delivered to Apoe−/− mice once per day, combined with or without Pg (107 CFU in 0.1 mL PBS) infection through intravenous injection once a week for 8 weeks. The untreated group underwent no injection.
At the terminal endpoint of the study, mice were sacrificed, and the heart and aortic arch specimens were harvested, followed by fixation in 4% PFA, or preservation at −80 °C for further detection. The reagents used are provided in Supplementary Table 5.
Histological staining
Serial 10-μm cross-sections were obtained from: (a) mouse aortic roots beginning at the first appearance of aortic valves, and (b) the same regions of rabbit aortic arches. Serial sections were allocated to consecutive slides. The whole sample was assessed at the same distance from the starting position. All parameters were quantified by multiple slides in each sample.
For analysis of atherosclerotic plaque instability, HE (S191003, Pinuofei), Oil red O (G1015-100ML, Servicebio), Masson (S191006, Pinuofei), and Prussian blue (P00129, Pinuofei) staining were applied. HE staining was employed to assess the area of the whole plaque and necrotic core. Lipid deposition was measured as the ratio of Oil Red O-positive area to total lesion area. Collagen content was expressed as the percentage of Masson-positive (blue) area within total lesion area. Fibrous cap thickness was assessed at its thinnest portion overlying most extensive necrotic core region. Prussian blue staining was employed to identify IPH. Quantification of stained area was assessed using NIH Image J software.
Immunofluorescence
Cellular composition of atherosclerotic plaques was analyzed by immunofluorescent staining. Plaque macrophages were detected by co-staining of CD45 antibody (60287-1-Ig, Proteintech) and F4/80 (28463-1-AP, Proteintech), or CD68 (A23205, Abclonal). Smooth muscle cells were detected by ACTA2 (14395-1-AP, Proteintech). T cells were detected by CD3 (176175-1-AP, Proteintech). Neutrophils were detected by co-staining of CD45 antibody and Ly6G (65078-1-Ig, Proteintech). Cy3-conjugated secondary antibodies (SA00009-1, Proteintech), Alexa 488-conjugated secondary antibodies (SA00013-2, Proteintech), and FITC-conjugated secondary antibodies (AS019, Abclonal) were applied for detection. T cell levels were expressed as the percentages of CD3-positive cells in total intraplaque cells. Other data were described as proportions of fluorescent area in the whole lesion.
MSR1 (A2401, Abclonal), p-MLKL (Ab196436, Abcam), FOXO3 (10849-1-AP, Proteintech), NOX2 (19013-1-AP, Proteintech) expression, and gingipain (CSB-PA338957LA01PQP, Cusabio) existence in macrophages were assessed by immunofluorescence staining. 4,6-diamidino-2-phenylindole (DAPI, C1002, Beyotime) was applied to nuclei visualization. IgG of the primary antibody was utilized as immunostaining control to validate antibody specificity and to distinguish background from genuine target staining. Comprehensive antibody details are tabulated in Supplementary Table 2.
Images were acquired by Nikon A1-Si confocal microscope. The fluorescent area was quantified utilizing NIH Image J software.
Fluorescence in situ hybridization (FISH)
Human arterial samples were sectioned into 5-μm-thick paraffin sections for FISH. FISH was conducted with a FITC-labeled oligonucleotide probe (5′-CAATACTCGTATCGCCCGTTATTC-3′, 5 mg/mL) targeting Pg ribosomal 16S rRNA sequences, followed by DAPI nuclear counterstaining.
Transmission electron microscope (TEM)
Fresh segments of aortic arch tissues were carefully isolated and minced into 2 mm3 size and immediately fixed in electron microscope fixator (G1124-100ML, Servicebio) at 4 °C for primary fixation. After rinsing with 0.1 M phosphate buffer, the specimens underwent post 2-h fixation in phosphate buffer containing 1% osmium acid, followed by dehydration in graded ethanol (50–100%). Subsequently infiltrate and embed the samples in epoxy embedding medium (90529-77-4, SPI), followed by polymerization at 60 °C for 48 h. Ultrathin sections were made with a Leica UC7 ultramicrotome and stained with uranyl acetate and lead citrate (30 min each). Sections were imaged on a JEM-1400Flash TEM at 80 kV voltage.
Determination of depletion efficiency of mononuclear phagocytes
At 72 h following intraperitoneal LC delivery, mice were anesthetized, and peripheral blood specimens (250 μL per mouse) were acquired via retro-orbital puncture into anticoagulated collection tubes. Erythrocytes were lysed through 5-minute incubation with ACK lysing buffer (Beyotime C3702) at a 1:3 sample-to-buffer ratio, and subsequently centrifuged at a low speed (500 × g, 5 min). After two PBS washes, leukocytes were immunolabeled using PE anti-mouse F4/80 (123109, BioLegend) and PerCP anti-mouse CD11b (101230, BioLegend). The samples were detected by flow cytometry.
Cell culture
RAW264.7 murine macrophages (from China Center for Type Culture Collection) and THP-1 human monocytes (from American Type Culture Collection) were cultured in high-glucose DMEM and RPMI-1640 medium, respectively, both supplemented with 10% heat-inactivated fetal bovine serum. All cells were maintained at standard incubation conditions (37 °C, 5% CO2, 90–95% humidity).
Cell apoptosis and necrosis assay
To detect intraplaque cell death, tissue sections were examined by TUNEL assay with in situ cell death detection kit (11684817910, Roche), complying with the instructions.
Apoptotic and necrotic cells in vitro were detected using Annexin V-FITC/PI Detection Kit (A211-01, Vazyme). The cells were collected, washed, and subsequently probed with PI and FITC-Annexin V for 15 min followed by flow cytometry analysis.
Cell oil red O staining
Macrophages in 24-well plates were stimulated with Pg (MOI = 100) or PBS in the presence of ox-LDL (60 μg/mL, YB-002, Yiyuan biotechnology) for 24 h. Whereafter, cells underwent fixation with 4% PFA, and were immersed in Oil Red O solution (G1015-100ML, Servicebio) for half an hour. After extensive PBS washing, cells were observed under the microscope. The lipid accumulation level was represented as the proportion of Oil Red O-positive cells relative to total cells.
Quantitative measurement of intracellular cholesterol
Macrophages in 12-well plates were stimulated with Pg (MOI = 100) or PBS in the presence of ox-LDL (60 μg/mL) for 24 h. Whereafter, macrophages were treated with pre-chilled RIPA lysis buffer and sonication. After centrifugation (12,000 × g, 10 min, 4 °C), supernatants were harvested for following detections. Protein concentrations were assessed using BCA assay (P0012S, Beyotime). The total cholesterol, cholesterol ester as well as free cholesterol concentrations were estimated using Total Cholesterol Assay Kit (BC1980, Solarbio) and normalized to total protein concentrations.
Cholesterol efflux assay
Macrophages incubated with 1 μg/mL BODIPY-cholesterol (HY-125746, MedChemExpress) for 6 h. Then cellular monolayers were rinsed with PBS and underwent 24-h incubation with 5 μg/mL apo-AI (HY-P72833, MedChemExpress) and 20 μg/mL HDL (YB-003, Yiyuan biotechnology) as cholesterol receptors in serum-free medium, along with Pg (MOI = 100) or PBS co-stimulation. Fluorescence intensity (Ex/Em 505/515 nm) was determined in both supernatant and cell lysate. Cholesterol efflux level was calculated as: medium fluorescence/ (cellular fluorescence + medium fluorescence). The reagents used are provided in Supplementary Table 5.
Lipid accumulation assay
After stimulation, macrophages in 12-well plates were stained with BODIPY 493/503 (10 μM, No. 25892, Cayman) for 30 min to detect ox-LDL uptake, or stained with Filipin (0.1 mg/mL, HY-N6716, MedChemExpress) for 30 min to detect free cholesterol content. After extensive PBS washing, cells were harvested, followed by flow cytometry analysis.
Viral infection, RNA interference, and plasmid transfection
For Msr1 gene silencing, macrophages were transduced with lentiviral vector (GV112, hU6-MCS-CMV-Puromycin, GENECHEM Biosciences) carrying Msr1-RNAi (NM_0011133269) at an MOI of 10. Following 24-h viral incubation in serum-free conditions, the old medium was substituted with complete growth medium containing puromycin (5 μg/mL) to select stably transduced cells.
For FOXO3 knockdown, macrophages were transfected with Foxo3-targeting siRNA oligonucleotides (GenePharma) using Lipo8000™ Transfection Reagent (C0533, Beyotime). Briefly, siRNA (0.1 μM) was mixed with Lipo8000 (6 μL) in serum-free medium (125 μL). After 15 min, the siRNA-Lipo8000 complex was added dropwise to cells. After 48-h incubation under standard culture conditions, cells were subjected to downstream assays.
The firefly luciferase reporter plasmids (PGL3-Basic, PGL3-Msr1, and their mutant/deletion variants) and the internal control Renilla luciferase plasmid pRL-TK were constructed by TsingKe Biological Technology (Wuhan, China). For transfection, cells were treated with a serum-free mixture containing the experimental reporter plasmids (500 ng), pRL-TK (125 ng), and Lipo8000 transfection reagent (1 μL). After 48-h incubation under standard culture conditions, cells were subjected to downstream assays.
Protein extraction and western blot
Macrophages were sonicated ten times using a high-intensity ultrasonic processor in ice-cold RIPA buffer (P0013B, Beyotime) containing fresh inhibitors of protease (P1005, Beyotime) and phosphatase (P1081, Beyotime). Following centrifugation (12,000 rpm, 4 °C, 10 min), supernatant was mixed with loading buffer (P0015, Beyotime) and denatured (95 °C, 5 min). Samples were detected using the BCA assay to quantify the protein concentrations (P0012S, Beyotime) prior to separation by SDS-PAGE (G2177, Servicebio) and transfer to 0.45 μm PVDF membranes.
Following 1-h blocking using 3% skim milk, membranes underwent successive incubation with primary antibodies (4 °C, 12 h) and HRP-conjugated secondary antibodies (23 °C, 60 min). Blots were visualized using ECL system. Antibody information is exhibited in Supplementary Table 2.
Detection of oxidative stress
For cultured macrophages, the ROS level was examined using the Total Oxygen Species Assay Kit (88-5930-74, Invitrogen). Briefly, following experimental treatments, cells were harvested and incubated with ROS Assay Stain for 60 min at 37 °C and 5% CO2. Fluorescence intensity was immediately measured by flow cytometry.
The ROS production within tissue sections from mouse aortic roots were measured using dihydroethidium (DHE, D7008, Sigma). Fresh segments of heart tissues were frozen in optimal cutting temperature compounds and sectioned into 10 μm-thick slices. Cryosections were incubated with 25 μg/mL DHE at 37 °C for 30 min. Cellular nuclei were probed using DAPI. Fluorescent images were taken by fluorescent microscopy (excitation 535 nm, emission 610 nm).
Quantitative real-time PCR (qRT-PCR)
Total RNA was extracted employing Trizol reagent (R401-01, Vazyme) complying with recommended procedures. For reverse transcription, 1000 ng of extracted RNA served as the template for cDNA synthesis, which was performed utilizing the HiScript II Q RT SuperMix for qPCR kit (22107, TOBOLO). qRT-PCR was then performed utilizing AceQ Universal SYBR qPCR Master Mix (22204, TOBOLO) on a StepOne Plus real-time PCR platform (Applied Biosystems). Gene expression levels were normalized to β-actin and quantified via the 2−ΔΔCt method. Primer information is exhibited in Supplementary Table 3.
Silver staining
207.9 μmol/L recombinant human FOXO3 (CSB-EP008836HU1, Cusabio) was incubated with 2.1 μmol/L RgpA (ab225982, Abcam), RgpB (CSB-EP310587EYA, Cusabio) or Kgp (CSB- EP690409PQP1, Cusabio) (37 °C, 60 min), followed by SDS-PAGE. Subsequently, the gels were processed using the Fast Silver Stain Kit (P0017S, Beyotime), complying with recommended procedures. All relevant reagents are detailed in Supplementary Table 5.
Chromatin immunoprecipitation (ChIP)
CHIP was performed utilizing the CHIP Kit (P2078, Beyotime) according to standardized protocols. Briefly, following formaldehyde crosslinking and cell lysis, DNA was sonicated into fragments less than 500 bp. Then, lysates were successively immunoprecipitated with 1 μg specific antibody (4 °C, 12 h), and protein A/G agarose beads (4 °C, 4 h). Antibody-chromatin complexes were washed sequentially with low salt, high salt, LiCl, and TE buffers, then eluted in elution buffer. Complexes were eluted and reversed in 200 mM NaCl (65 °C, 5 h). Purified DNA was obtained after RNase/proteinase K treatment and column purification. qRT-PCR was conducted using primers targeting the predicted FOXO3 binding sites on the Msr1, MSR1, Tnfsf10, Cat, and Casp6 promoters. The DNA samples were detected for further 1% agarose gel electrophoresis. The CHIP-PCR primer information is exhibited in Supplementary Table 4.
Luciferase reporter assay
Macrophages were seeded (105 cells per well) in 24-well plates following transfection with either plasmids or an empty vector. After 48-h transfection, cells were treated with ox-LDL, combined with or without Pg or KDP136 stimulation for 24 h.
Following stimulation, cells were harvested and lysed for subsequent luciferase activity quantification. Firefly and Renilla luciferase activities were sequentially detected using the Dual-Luciferase Reporter Assay System (Promega). To assess relative Msr1 promoter-driven transcriptional activity, the luminescence signal was normalized by calculating the Firefly luciferase (Fluc) to Renilla luciferase (Rluc) ratio.
Co-immunoprecipitation (Co-IP)
Following stimulation, macrophages were lysed with Cell lysis buffer (P0013, Beyotime) containing fresh protease inhibitors (P1005, Beyotime) at 4 °C for 2 h. After centrifugation to remove debris, the lysates were successively incubated with specific primary antibodies (4 °C, 12 h) and with protein A agarose beads (PR40023, Proteintech) (4 °C, 4 h). Immunocomplexes-beads mixture was then washed with binding buffer for four times, and eluted in protein loading buffer (95 °C, 5 min). Samples were then subjected to western blotting analysis. Antibody information is exhibited in Supplementary Table 2.
Proteomic analysis
Macrophages in 12-well plates were treated with Pg (MOI = 100) or PBS combined with ox-LDL (60 μg/mL) for 1 day. Whereafter, cells were collected and subjected to proteomic analysis. Protein was extracted as described before and underwent trypsin digestion. Then, peptides were subjected to TMT labeling using TMT reagents (Thermo Fisher Scientific) and analyzed through LC-MS/MS with EASY-nLC 1200 system, coupled to Q Exactive™ HF-X mass spectrometer using data-dependent acquisition (DDA) mode (350–1600 m/z at 60,000 resolution). Data were managed with MaxQuant search engine (v.1.6.15.0) against UniProt mouse FASTA database (Mus_musculus_10090_SP_20220107.fasta). FDR was adjusted to less than 1%. Differential proteins between Pg group and PBS control group with P value < 0.05 were regarded as statistically significant and identified as upregulated or downregulated proteins with a fold-change (Pg/PBS) > 1.15 or <0.87.
The raw mass spectrometry proteomics data have been deposited in the ProteomeXchange Consortium (https://proteomecentral.proteomexchange.org) via the iProX partner repository67,68 with the dataset identifier PXD048371.
Statistical analysis
All statistical analyses were processed via GraphPad Prism 9.0. Shapiro–Wilk test was applied to examine data normality. For data with normal distribution, unpaired two-tailed Student’s ttest was used for comparisons between two groups. One-way ANOVA, combined with Tukey post-hoc tests and repeated measure ANOVA, combined with Bonferroni post-hoc tests, were applied for multiple group comparisons. For non-normally distributed data, Mann–Whitney U test was used for comparisons between two groups, and Kruskal–Wallis test combined with Dunn’s multiple comparison tests was used for multiple group comparisons. For comparisons involving categorical variables, Fisher’s exact test was used. All results were described as mean ± standard error of the mean (SEM), or mean ± standard deviation (SD). n represented animal numbers or numbers of independent experiments in each experiment, as detailed in the figure legends. Values of p < 0.05 represented statistically significant difference.
