NLRP3 inflammasome-modulated angiogenic function of EPC via PI3K/ Akt/mTOR pathway in diabetic myocardial infarction

Figures

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  Fig. 1. Impaired cardiac function and promoted left ventricular remodeling after permanent myocardial ischemia in HFD/STZ-induced diabetic mice. (A) a diagram of the experimental protocol. (B) Short-term survival curve after myocardial infarction and the indicated treatments (n = 10–12 mice/group). (C) Representative short-axis M-mode echocardiograms of the left ventricle in the control-Sham, HFD/STZ-Sham, control-MI, and HFD/STZ-MI groups. Left ventricular function was assessed by measurements of ejection fraction (D), LV fractional shortening (E), end diastolic volume (F), end systolic volume (G), and at day 7 and day 21 after MI (n = 6 mice/group). (H) Representative images and quantitative infarct size in Masson’s trichrome stained mice hearts on day 21 after MI (n = 6 mice/group). (I) Representative endothelial CD31 staining at the infarction border zone sections. *p < 0.05, **p < 0.01, ***p < 0.001 vs. Control-sham, # p < 0.05, ## p < 0.01, ### p < 0.001 vs. Control-MI group. Similar results were obtained from three independent experiments

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  Fig. 2. Reduced EPC mobilization and activated NLRP3 inflammasome in HFD/STZ-induced diabetic mice. (A) a diagram of the experimental protocol. (B) FACS analysis on peripheral blood mononuclear cells for EPC mobilization in groups. (C) The bar graph showed that EPC mobilization was impaired in HFD/STZ-induced diabetic mice as compared with control mice (n = 6 mice/group). (D) Nlrp1, Nlrp3, Nlrp6, Nlrc4 and Aim2 mRNA expression (RT-PCR) in flow cytometry-sorted EPC were quantified at 3 days after MI. (E) Nlrp3, Caspase1, and Il-1b mRNA expression (RT-PCR) in flow cytometry-sorted EPC were quantified at 3 days after MI. mRNA expression normalized to Actb and depicted as fold change versus control. (F) a diagram of the matrigel plug protocol. (G) Representative HE photographs of matrigel plugs removed from mice 10 days after injection (n = 6 mice/group). The relative amounts of hemoglobin extracted from the excised matrigel plugs and the number of blood vessels in matrigel plugs (n = 6 mice/group). (H) Relative NLRP3, Caspase1 p20, and IL-1β protein expression levels in EPC extracted from the excised matrigel plugs (n = 3 mice/group). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.…

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  Fig. 3. Intramyocardial injectionNlrp3 knockout EPC improved cardiac function and restrained left ventricular remodeling after permanent myocardial ischemia in HFD/STZ-induced diabetic mice. (A) a diagram of the transplanted experimental protocol. (B) Representative images showing that the fluorescence signal observed in the heart of myocardial ischemia mice on days 1, 7 and 21 after injection of Dil-labeled EPC. (C) Fasting glucose, oral glucose tolerance test and HbA1c in HFD/STZ-induced diabetic myocardial infarction mice followed by intramyocardial injection of EPC. (D) Representative short-axis M-mode echocardiograms of the left ventricle at day 21 in the control and HFD/STZ-induced diabetic groups. (E) Left ventricular function after injection of Nlrp3 knockout EPC was assessed by measurements of ejection fraction, LV fractional shortening, end diastolic volume, and end systolic volume at day 7 and day 21 after MI (n = 6 mice/group). (F) Left ventricular function was assessed by measurements of ejection fraction, LV fractional shortening, end diastolic volume, and end systolic volume at day 21 after MI (n = 6 mice/group). (G) Representative endothelial CD31 staining at the…

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  Fig. 4. Regulated angiogenesis function of EPC by NLRP3 inflammasome in response to HG or AGEs. (A) Western blot analysis of NLRP3, Caspase1, and IL-1β cleavage in EPC from WT or Nlrp3−/− mice following stimulation with HG (30 mM) or AGEs (200 µg/ml) for 72 h. The supernatant was collected directly after a 72-hour incubation period with high glucose and AGEs. (B) EPC from wild-type mice were transfected with Vector or Nlrp3. Western blot analysis of NLRP3, Caspase1, and IL-1β cleavage in EPC transfected with Vector or Nlrp3 following stimulation with HG (30 mM) or AGEs (200 µg/ml) for 72 h. (C) ELISA analysis of IL-18, and IL-1β in supernatants of EPC from WT or Nlrp3−/− mice and EPC transfected with Vector or Nlrp3 following stimulation with HG (30 mM) or AGEs (200 µg/ml) for 72 h. (D) Wound healing of EPC response to HG (30 mM) or AGEs (200 µg/ml) in vitro. (E) Migration assay with EPC treated with HG (30 mM) or AGEs (200 µg/ml) was performed. (F) The angiogenic function of EPC was evaluated by tube formation assay. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Representative results from three biologically independent experiments

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  Fig. 5. Regulated proliferation function of EPC by NLRP3 inflammasome in response to HG or AGEs. (A) Flow cytometry analysis was conducted on EPC following stimulation with HG (30 mM) or AGEs (200 µg/ml) for 72 h. The representative pattern of the cell cycle distribution of EPC in synchrony after stimulation. The representative pattern of the cell cycle distribution of EPC in synchrony and after stimulation. (B) Representative images of BrdU incorporation status after stimulation with HG (30 mM) or AGEs (200 µg/ml) for 72 h in EPC. Images were generated by merging the DAPI and BrdU channels. (C) Clone formation assay results showed the effects of NLRP3 inflammasome on the proliferation of EPC. (D) Senescence-associated beta-galactosidase staining of EPC following stimulation with HG (30 mM) or AGEs (200 µg/ml) for 72 h. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Similar results were obtained from three independent experiments

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  Fig. 6. CEBPB initiated the transcription ofNlrp3by directly binding to the promoter region ofNlrp3. (A) The volcano map of DEGs in EPC at the transcriptional level following stimulation with HG (30 mM) or not for 72 h (∣log2fold change∣ > 1 and FDR < 0.05). Venn diagram based on the overlapping DEGs between upregulated genes and mouse transcription factor. Differentially expressed transcription factors and adjust P.value in HG vs. Control group. (B After transfection with various transcription factors, luciferase reporter gene assays using the Nlrp3 promoter were performed in EPC. (C) Relative NLRP3 and CEBPB protein levels in EPC following stimulation with HG (30 mM) or AGEs (200 µg/ml) for 72 h. (D) Correlation between NLRP3 levels and CEBPB levels in EPC. (E) Western blot of nuclear extracts obtained from EPC following stimulation with HG (30 mM) or AGEs (200 µg/ml) for 72 h was performed to investigate the expression of CEBPB. Each density of CEBPB was normalized with that of LAMIN B. (F) Representative immunofluorescence imaging of CEBPB nuclear translocation after stimulation with HG (30 mM) or AGEs (200 µg/ml) for 72 h. (G) Luciferase reporter assay in EPC using Nlrp3 promoter…

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  Fig. 7. USP14 interacted with NLRP3 and inhibited NLRP3 protein degradation. (A) The expression of Nlrp3 mRNA at 0,4,8 and 12 h post-stimulation was determined by qRT-PCR and normalized to the 18 S rRNA. (B) Immunoblot analysis of extracts from EPC stimulated with HG (30 mM) for 72 h, followed by treatment with cycloheximide for various times (CHX, 100 ug/mL). NLRP3, PYCARD, and Caspase1 expression levels were quantitated by measuring band intensities and normalized to ACTB. (C) Heatmap showing the expression of USP family genes identified in control EPC vs. HG (30 mM) treated EPC. (D) MYC-Nlrp3 was transfected in EPC, as well as graded amounts of FLAG-Usp14, FLAG-Usp11 and FLAG-Usp12. The protein level of NLRP3 were detected by western blot. MYC-Caspase1, and MYC-Pycard were transfected in EPC, as well as graded amounts of FLAG-Usp14. The protein level of Caspase1, and PYCARD were detected by western blot. (E) EPC was stimulated with HG (30 mM) or AGEs (200 µg/ml) for 72 h, then fixed and incubated with a secondary antibody. Colocalization between USP14 and NLRP3 was examined by confocal microscopy. Confocal imaging results are representative of three independent experiments. (F)…

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  Fig. 8. CEBPB and USP14 regulated cardiac Function by modulating NLRP3 inflammasome in vivo. (A) Short-term survival curve after myocardial infarction and the indicated treatments. (B) Representative short-axis M-mode echocardiograms of the left ventricle at baseline and day 21 in the HFD/STZ-induced diabetic mice with myocardial infarction groups. Left ventricular function was assessed by measurements of ejection fraction, LV fractional shortening, end diastolic volume, and end systolic volume. (C) Representative endothelial CD31 staining at the infarction border zone sections. (D) Representative images and quantitative infarct size in Masson’s trichrome stained mice hearts on day 21 after MI (n = 6 mice/group). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. These experiments were repeated independently at least three times with similar results

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  Fig. 9. NLRP3 inflammasome activation inhibited angiogenesis via PI3K/Akt/mTOR pathway in EPC. (A) GO enrichment analysis of the known molecular function in wild-type and Nlrp3 knockout EPC stimulated with high glucose (30mM) for72h. (B) KEGG enrichment analysis of the known oncogenic or metabolic pathways in wild-type and Nlrp3 knockout EPC stimulated with high glucose (30mM) for72h. (C) Western blot analysis of p-MTOR, MTOR, p-AKT, AKT, p-PI3K, PI3K protein levels in EPC pretreated with MCC950 (10µM, 8µM, 4µM, 2µM, 1µM) for 6 h in response to HG (30 mM). (D) Western blot analysis of p-MTOR, MTOR, p-AKT, AKT, p-PI3K, PI3K protein levels in EPC pretreated with MCC950 (10µM, 8µM, 4µM, 2µM, 1µM) for 6 h in response to AGEs (200 µg/ml). (E) Western blot analysis of p-MTOR, MTOR, p-AKT, AKT, p-PI3K, PI3K protein levels in EPC pretreated with shNlrp3, LY294002 (20µM) for 12 h, and 3-methyladenine (5 Mm) for 12 h. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. Control. These experiments were repeated independently three times with similar results