Animals

Eight-week-old wild-type (C57BL/6J, WT) and Nlrp3 knockout (C57BL/6J, Nlrp3 KO) male mice were procured from Nanjing GemPharmatech company. All experimental protocols were approved by the ethic committee of China Pharmaceutical University, following the Guidelines of Animal Experiment set by the Bureau of Sciences and Techniques of Jiangsu Province, China [NO. SYXK2007-0025].

Preparation of the HFD/STZ-induced diabetic mice model

Adult C57BL/6J male mice were divided into two groups: control, and untreated diabetic group. During the first 6 weeks, mice in the untreated diabetic group were given a high-fat diet (HFD, 10% sucrose, 10% lard, 10% sugar, 5% egg yolk powder, 0.5% cholesterol, 64.5% basic feed) purchased from Qinglong Mountain Laboratory Animals Ltd. Control mice received only a regular diet. At the beginning of the 7th week, HFD-feeding mice were intraperitoneally injected three times with streptozotocin (STZ, 35 mg/kg body weight, dissolved in pH 4.5 citrate buffer, Sigma, N572201) to induce partial loss of pancreatic β-cells as described previously. Control mice only received an equivalent volume of citrate buffer. After 1 week of STZ injection, mice were identified as diabetic mice with fasting blood glucose (FBG) levels reaching 16.7 mM (Supplementary Fig. 1A). We established the HFD/STZ-induced diabetic mice model by assessing multiple indicators including insulin levels (Supplementary Fig. 1B), HbA1c levels (Supplementary Fig. 1C), Oral Glucose Tolerance Test (OGTT) (Supplementary Fig. 1D), and Insulin Tolerance Test (ITT) (Supplementary Fig. 1E).

Myocardial infarction and cell therapy

Myocardial infarction was induced by ligation of the left coronary artery as previously described [22, 23]. Animals are induced with 5% isoflurane in the induction chamber, then intubated and mechanically ventilated with 1–3% isoflurane to maintain the depth of surgical anesthesia during the procedure. Surgical pain was reduced by preincisional subcutaneous injection of buprenorphine 0.05 mg/kg. A left open-heart procedure is performed in the fourth intercostal space of the mouse, followed by a pericardiotomy. The left coronary artery is ligated with a 6 − 0 circular suture, and ST-band elevation is observed on the ECG. EPC were transplanted to the border area of myocardial infarction by injecting at three different but adjacent sites using a syringe. The number of cells injected at each site was 4 × 105, and the PBS group was injected with the same volume of PBS. The heart is rapidly returned to the thorax, which is closed and the mice are allowed to recover using assisted mode ventilation. Pulmonary atelectasis was prevented by generating positive end-expiratory pressure at the end of the procedure. Sham control animals underwent the same surgical procedure in the absence of coronary occlusion. The surgical mortality rate in HFD/STZ-induced diabetic mice after myocardial infarction is approximately 22%, which suggests that our surgical approach is feasible. For euthanasia, mice were placed in a sealed chamber and were euthanized by CO2 asphyxiation, followed by cervical dislocation.

Echocardiography

Echocardiography was performed using a 2% isoflurane anesthesia delivered through a Viking Medical system (Medford, NJ) in mice. Transthoracic two-dimensional M-mode echocardiograms were obtained with the Vevo 3100 equipped with a 30 MHz transducer (Visual Sonics, Toronto, Canada). Echocardiographic studies were conducted before myocardial infarction (baseline) and at 1, 7, and 21 days post-myocardial infarction under anesthesia using a mixture of 1.5% isoflurane and oxygen (1 L/min). The left ventricular (LV) internal diameter was measured in the short-axis view recorded in M-mode; ejection fraction (EF) and fractional shortening (FS) were calculated using the formulas previously described [24].

Bone marrow cell isolation and EPC culture

Briefly, bone marrow mononuclear cells were isolated from mice by density gradient centrifugation with Histopaque-1083 at room temperature and depleted of macrophages by allowing attachment to uncoated plates for 1 h. The unattached cells were removed and plated on 6-well type I collagen-coated tissue culture plates and cultured in phenol red-free endothelial cell basal medium-2 (EBM-2, Clonetics) supplemented with 10% FBS, vascular endothelial growth factor (VEGF)-A, fibroblast growth factor-2, epidermal growth factor, insulin-like growth factor-1, ascorbic acid, and antibiotics. Cells were cultured at 37 °C with 5% CO2 for 48 h before changing media to remove non-adhered cells by washing with PBS. Media was changed every 2 days until colonies appeared after 3 weeks. These EPCs were utilized in subsequent in vivo and in vitro experiments. Similarly, Nlpr3 knockout EPC were isolated from bone marrow mononuclear cells of Nlrp3 knockout mouse according to the above experimental procedure.

FACS sorting

To investigate whether diabetic condition affects MI-induced mobilization of BM-EPC into the circulation, we performed myocardial infarction (MI) in control and HFD/STZ induced diabetic mice and assessed EPC mobilization (Sca-1+ Flk1+ cells) by FACS analysis on peripheral blood mononuclear cells. Freshly isolated mononuclear cells from peripheral blood by histopaque-1083 were stained with Phycoerythrin(PE)-conjugated Rat anti-mouse stem cell antigen 1 (Sca-1) and Allophycocyanin (APC)-Rat conjugated anti-mouse fetal-liver kinase 1 (Flk1) antibodies (BD Pharmingen Inc.) in 1% BSA. Isotype-matched IgG antibodies were used as negative controls. Quantitative fluorescence analyses were performed with a CytoFLEX cytometer (Beckman Coulter, Inc.) and Flow-Jo Software (Tree Star, Inc.); 50,000 events were counted for each sample.

Plasmid construction and transfection

MmNlrp3-coding (Gene ID:216799), MmCaspase1-coding (Gene ID:12362), and MmPycard-coding (Gene ID:66824) sequence were amplified from EPC by touch-down PCR and cloned into pCMV-Myc-N (635689, Clontech). MmUsp14-coding sequence (Gene ID:59025) was cloned into p3XFLAG-CMV-14 (Sigma). The deletion and truncation sequence of Nlrp3 were amplified from EPC by touch-down PCR and cloned into pCMV-Myc-N. pCDH-CMV-MCS-EF1-copGFP-T2A-Puro-Nlrp3 was generated by subcloning the MmNlrp3-coding sequence into the pCDH-CMV-MCS-EF1-copGFP-T2A-Puro (CD513B-1, SBI), and the empty pCDH-CMV-MCS-EF1-copGFP-T2A-Puro was used as a control. Plvx-mcherry-Usp14 was generated by subcloning the MmUsp14-coding sequence into the plvx-mcherry-N1 (632562, Clontech), and the empty plvx-mcherry-N1 was used as a control. In addition, the shRNA targeting Usp14 and Nlrp3 mRNA sequence was introduced into pSIH1-H1-copGFP-T2A-Puro vector (System Biosciences) under the control of the H1 promoter. The Nlrp3 shRNA targeting sequence was 5’- CCGGCCTTACTTCAATCTGTT-3’. The Usp14 shRNA targeting sequence was 5’-CCTCCGAAAGAGATTAAGTAT-3’. Sh-NC was constructed (5’-GAAGCAGCACGACTTCTTC-3’) with no significant homology to any mammalian gene sequence. All constructed plasmids are verified by DNA sequencing. Plasmids were transiently transfected into EPC with Lipofectamine 2000 Transfection Reagent (Invitrogen, USA) according to the manufacturer’s protocol.

Lentivirus production

To make lentiviral particles, HEK 293T cells were grown in a 100 mm cell culture dish in DMEM media without antibiotics. When cells were 70 ~ 80% confluent, 10 µg lentiviral transfer plasmid, 5 µg psPAX2, and 2.5ug pMD2.G were co-transfected into HEK-293T cells with Lipofectamine 2000 Transfection Reagent (Invitrogen, USA) according to the manufacturer’s protocol. Lentiviral particles were harvested at 48 and 72 h post-transfection and concentrated with L-80XP Super Speed Centrifuge (BECKMAN). The lentiviruses were subsequently used to infect EPC.

Luciferase assay

Cells were plated in 96-well plates 24 h before transfection. Plasmids containing different fragments of the Nlrp3 promoter were generated by subcloning the Nlrp3 promoter sequence into the pGL4.17 (Promega) vector. All plasmids containing different fragments of the Nlrp3 promoter were co-transfected with a control Renilla luciferase plasmid (pRL-TK). The ratio of the experimental plasmid to the control plasmid was set at 50:1. Luciferase activity was measured using the Duo-Lite Luciferase Assay System according to the manufacturer’s instructions (DD1205, Vazyme). Briefly, at 24–48 h after transfection, cell lysates were prepared by incubating the cells with the same volume of Duo-Lite Luciferase reagent for 10 min at room temperature. The firefly luminescence signal was detected with Promega GloMax 20 according to the manufacturer’s instructions. After this, duo-Lite Stop reagents are added for the detection Renilla luminescence signal. Firefly luminescence signal in transfected cells was normalized to Renilla luminescence signal.

Chromatin immunoprecipitation assay

The chromatin immunoprecipitation (ChIP) assays were performed according to the standard ChIP protocol (Millipore). Briefly, EPC was harvested and fixed with 1% formaldehyde for 10 min at room temperature, then crosslinking was quenched with 2-M glycine for 5 min. The chromatin was sonicated until the DNA fragments were 500 bp in size after nuclei isolation. After sonication, 2% percent of the sonicated chromatins were used as input control, and the rest sonicated chromatins were incubated with specific antibodies. Chromatin fragments containing DNA-protein were precipitated by using 5 µl of anti-C/EBPβ (Proteintech) or 5 µl of anti-IgG (Beyotime Biotechnology). The immunoprecipitated complex was washed with low salt immune complex wash buffer, high salt immune complex wash buffer, LiCl immune complex wash buffer, and TE buffer. The DNA was extracted and purified by DNA Extraction Mini Kit (Vazyme).

The enrichment of specific DNA sequences was examined by PCR using specific primer pairs of the different sites of Nlrp3 promoters. Primers were used as follows:

Site1-F 5′-TCCATCCAGATGAGTAACTGCCAATCC-3′.

Site1-R 5′-GGGTCACTGTGACACTGGAGAC-3′.

Site2-F 5′-CCAGATTTCTGGAGACCAACCTAGT-3′.

Site2-R 5′-AGCCAGACTCAGGAAGACAGGAG-3′.

Site3-F 5′-GCTCATCCTCCTATGATGGAGTTG-3′.

Site3-R 5′-GGCTCAAAGAAGCCACTAATGACC-3′.

Nuclear protein extraction

The nuclear protein extraction was performed using the Nuclear and Cytoplasmic Protein Extraction Kit (P0027, Beyotime, China) according to the manufacturer’s instructions. Cells were collected in PBS by scraping from culture flasks and washed twice with cold PBS. The supernatant was removed and the cell pellet was collected into a prechilled microcentrifuge tube. The cell pellet was resuspended with 200 µL Buffer A containing 1 mM PMSF and incubated on ice for 15 min. Then 10 µL buffer B was added and the mixture was vortexed for 5 s for 1 min, followed by centrifugation at 12,000 g, 4 °C for 5 min. The supernatant was removed and the precipitation was resuspended in a 50 µL nuclear protein extraction reagent containing 1 mM PMSF. The mixture was vortexed for 30 min and centrifugated at 12,000 g, 4 °C for 5 min. The supernatant was collected as nuclear protein and analyzed by Western blotting assay.

Co-immunoprecipitation

Cells were washed twice with cold PBS and collected by scraping from culture flasks. Collected cells were lysed in IP lysis buffer containing 1 mM PMSF and centrifuged at 14,000 g, 4 °C for 10 min. The supernatant was transferred into a microcentrifuge tube and incubated with 2 µg primary antibody (Beyotime Biotechnology) overnight at 4 °C. Then the mixture was incubated with 20 µl fully resuspended Protein A + G Agarose for 3 h. Protein A + G Agarose was washed five times with IP buffer. The immunoprecipitates were eluted by boiling with 1% (wt/vol) SDS buffer and analyzed by Western blots assay.

Protein preparation and western blots

Supernatant protein: The precipitation of supernatant proteins was performed using the methanol/chloroform method. Initially, culture media were centrifuged at 2000 × g for 10 min to pellet the cells and cell debris. Subsequently, 600 µL of the supernatant was transferred to a new tube, followed by the addition of 600 µL of methanol and 150 µL of chloroform. The samples were thoroughly mixed and centrifuged at 12,000 × g for 10 min. The upper phase was discarded, and 600 µL of methanol was added to each sample. After mixing well, the samples were centrifuged again at 12,000 × g for 10 min. The protein contents were deter-mined using Bradford reagent (Bio-R ad protein assay kit) and aliquots normalized to equal quantities before loading. The supernatants were removed, and the remaining protein pellets were dried at 55 °C, then resuspended in 2× SDS loading buffer. Finally, the samples were boiled at 98 °C for 10 min until fully dissolved.

Matrigel plug: Tissue samples from mice, embedded in Matrigel, were homogenized using a homogenizer and subsequently lysed in RIPA buffer. Protein concentrations were determined using the Bradford assay, with 60 µg of protein loaded into each well for analysis.

Cell lysates: EPC was lysed in SDS-RIPA lysis buffer and protease inhibitor cocktail. After 15 min centrifugation at 12,000 × g at 4 °C, protein concentrations in the lysates were collected and eluted by boiling with 1% (w/v) SDS sample buffer. Equal amounts of proteins were separated by SDS–PAGE and then were transferred onto PVDF membranes for immunoblot analysis. After membranes were blocked with 5% skim milk for 1.5 h at room temperature, and the blocking buffer was changed to 5% BSA for phosphorylation, they were probed with various primary antibodies overnight at 4 °C. After washing for three times, the membranes were incubated with secondary antibodies for 1.5 h at room temperature and visualized with chemiluminescent systems with LumiGlo and Peroxide. The intensity of protein bands was measured using ImageJ image analysis software (NIH, Littleton, CO, USA).

Immunofluorescence microscopy

EPC was plated on glass coverslips in 24-well plates and fixed with 4% paraformaldehyde for 15 min. Cells were permeabilized with 0.3% Triton-X 100 in phosphate buffer saline (PBS) and blocked with 5% goat serum for 1 h. Then the cells were incubated with anti-USP14 and anti-Nlrp3 diluted in Immunol Staining Primary Antibody Dilution Buffer (Beyotime) at 4 °C overnight. Next, the cells were stained with secondary Antibodies, and nuclei were stained with DAPI (Solarbio, Beijing, China). The fluorescent images were photographed by a Laser scanning confocal microscope (ZEISS Scope. A1, Germany) and the images were processed by ZEN blue 2.3 software (Carl Zeiss, Germany). Pearson’s coefficient which represents colocalization was calculated by Image J software (NIH, Littleton, CO, USA).

ELISA

The concentrations of mouse IL-1β (cat. EK201B), and mouse IL-18 (cat. EK218) were measured using ELISA kits (lianke Biotech Company Ltd., Hangzhou, China) according to the manufacturer’s instructions.

Angiogenesis assay

The angiogenic capability of EPC was determined by matrigel tube formation assay. Briefly, 96-well plates were coated with matrigel (50 µL/well; BD Biosciences) at 37 °C for 30 min. Then, 3 × 104 EPC were plated on matrigel per well and incubated at 37 °C with 5% CO2 for 6 h. Six hours post-treatment, the digital images of the tubes were photographed with a phase-contrast microscope. The total tube length was counted in each well by Image J software.

Matrigel plug assay

The angiogenesis model was established using Matrigel (BD) implants in mice. Specifically, 500µL of Matrigel, with 5 × 105 EPC, was injected subcutaneously into the mid-dorsal region of 8-week-old male mice. The injected Matrigel quickly formed a single, solid plug. After 10 days, the Matrigel plugs were surgically removed from the mice, ensuring the exclusion of connective tissues. The neovascularization of Matrigel plugs was quantified by adding 4 ml of Drabkin’s reagent to 20 ml of well-homogenized neovascularized Matrigel. Following thorough mixing, the absorbance was measured at a wavelength of 540 nm using a spectrophotometer to estimate hemoglobin levels. Hemoglobin concentration was calculated using the formula: Hb (g/dl) = (absorbance of sample / absorbance of standard) × concentration of standard. The excised Matrigel was then embedded in paraffin and sectioned for HE analyses. The infiltrated vessels were counted in three Matrigel H&E-stained sections and expressed as the mean number.

Bioinformatics analyses

RNA sequencing was performed on an Illumina HiSeq 4000 platform of aptbiotech Co., Ltd. (Shanghai, China) to evaluate the differential gene expression profiles in EPC. The gene expression level was normalized by using the EdgeR method. The criterion for significant DEGs (differentially expressed genes) was set to at least a 2-fold change in gene expression, with an adjusted P-value < 0.01. The DEGs were enriched by KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways with the GSEA (gene set enrichment analysis) method in the ClusterProfiler package.

Statistical analysis

Data were presented as mean ± standard error mean (S.E.M.). Statistical analysis was performed with IBM SPSS Statistics 20 software. The Independent-Samples t-test was used to detect significant differences between the two groups. Significant differences between and within multiple groups were examined using ANOVA for repeated measures, followed by Duncan’s multiple-range test. In addition, for the sample groups where n = 3, Mann-Whitney test was employed. Kaplan–Meier survival curve was used for survival analysis, and the log-rank test was used to determine the statistical significance. P < 0.05 was considered statistically significant.