Macrophage exosome-modified Mn/Se nanoheterostructure induces post-infarction cardiomyocyte regeneration and immunometabolic microenvironment repair

Figures

• 

  Fig. 1. Schematic illustration of the therapeutic mechanisms of PMS@ME-mediated MI therapy. (A) Schematic of the PMS@ME fabrication. (B) After intravenous injection, PMS@ME is effectively recruited and accumulated in the injured cardiac tissue due to the adhesion targeting of M1 macrophage-derived exosomes, and inhibits the Hippo signaling pathway to promote cardiomyocyte proliferation. Specifically, PMS@ME reduces the phosphorylation of the complex of MST1/2 (Mammalian Sterile 20-like Kinase 1/2) and SAV1 (Salvador Family WW Domain Containing Protein 1) and LATS1/2 (Large Tumor Suppressor Kinase 1/2), thereby promoting YAP (Yes-associated Protein 1) translocation into the nucleus for binding to TEAD (TEA Domain Transcription Factor). Conversely, YAP failing to translocate into the nucleus is degraded either after binding to 14-3-3 (14-3-3 Protein Family) or directly. (C) PMS@ME therapy remodels the immune microenvironment, including inhibition of M1 macrophage and neutrophil infiltration, as well as increasing the number of M2 macrophages and Tregs. (D) PMS@ME improves cardiomyocyte survival status, mitochondrial metabolism, and extracellular microenvironment repair

• 

  Fig. 2. Validation and characterization of PMS@ME. (A) Transmission electron microscopic (TEM) images of PDA-MnSe (PMS). Scale bars, 200 nm and 50 nm. (B) Energy dispersive X-ray spectroscopy (EDS) mapping of C, N, O, Mn, and Se of PMS. Scale bars, 50 nm. (C) Western blot analysis of CD44 and CD11b expression from membranes of bone marrow-derived macrophages (BMDM-M) and membranes of inflammatory-stimulated M1 macrophages (IM-M). (D) Western blot analysis of Calnexin, CD63, CD81, and CD9 in IM-M and exosomes derived from inflammatory-stimulated M1 macrophages (ME). (E) TEM images of PMS@ME, after negative staining with uranyl acetate. Scale bars, 50 nm. (F) Fluorescence colocalization of PMS and ME in PMS@ME, and Control represents a direct mixture of FITC-labeled PMS and Dil-labeled ME. Scale bar, 10 μm. (G) Western blot of CD44 and CD11b expression in IM-M, ME, and PMS@ME. (H) Western blot of vascular cell adhesion molecule-1 (VCAM-1) and Integrin β1 expression in the hearts of wild-type mice and MI mice. (I) Size distribution of MnSe, PMS, and PMS@ME. (J) XRD spectra of MnSe and PMS. (K) Fourier transform infrared spectroscopy (FTIR) of PDA, MnSe, and PMS. (L, M) XPS spectra of MnSe…

• 

  Fig. 3. ROS-scavenging properties of PMS. (A) SOD-like activities to scavenge •O2⁻ by UV-vis utilizing a SOD kit in the Control, PDA, MnSe, and PMS groups. (B) Electron spin resonance (ESR) curves of scavenging hydroxyl radical (•OH) in different groups. (C) ESR curves of superoxide anion (•O2⁻) in different groups. (D) The concentration of dissolved oxygen produced by different groups. (E) H2O2 scavenging rate after treatment with PDA, MnSe, or PMS (n = 3). Data are presented as mean ± SD. P values are calculated using one-way ANOVA with Tukey’s corrections. (F) SOD-like activities of different concentrations of PMS by UV − vis utilizing a SOD kit. (G, H) ESR curves of scavenging •OH and •O₂⁻ with different concentrations of PMS. (I) The concentration of dissolved oxygen changes with different solutions of PMS at different time points

• 

  Fig. 4. PMS@ME targets damaged heart, improves cardiac function, and reduces infarct size. (A, B) Ex vivo fluorescence images and quantitative analysis of hearts harvested from different groups. Control: Sham mice injected intravenously with Cy5.5-labeled PMS. PMS/PMS@ME: MI mice injected intravenously with Cy5.5-labeled PMS/PMS@ME (n = 3). (C, D) Representative heart M-mode images and analysis of left ventricular ejection fraction (EF), left ventricle fractional shortening (FS), left ventricular end-systolic volume (LVESV), and left ventricular end-diastolic volume (LVEDV) at days 3 and 14 after MI surgery (n = 5). (E) Description of the anatomical positions of α, β, and γ sections shown in panel F. (F) Representative MRI LGE sequence images of myocardial infarct area at postoperative days 4 and 14 in different groups (n = 5). (G) Representative MRI CINE sequence images of cardiac cycle at postoperative day 4 (n = 5). (H) Representative Masson staining images and quantitative analysis of the infarct zone on day 14 post-surgery in different groups. (n = 5). Scale bar, 1 mm. Data are presented as mean ± SD for B, D, and H. P values are calculated by one-way ANOVA with Tukey’s post-hoc test

• 

  Fig. 5. PMS@ME promotes ECM deposition and angiogenesis, and inhibits cardiomyocyte hypertrophy and death. (A) Immunofluorescence analysis of collagen I, collagen III, and α-SMA expression in the infarct border zone of different groups at postoperative day 14 (n = 5). Scale bar, 50 μm. (B, C) Immunofluorescence images and quantitative analysis of CD31 expression in the infarct border zone of different groups at postoperative day 14 (n = 5). Scale bar, 50 μm. (D, E) Representative WGA staining images and quantification in the infarct border zone at postoperative day 14 (n = 5). Scale bar, 25 μm. (F, G) Western blot analysis of apoptosis-related proteins Bax and Bcl-2 in the myocardial ischemic tissues after different treatments on postoperative day 3 (n = 5). (H) Co-staining of cTnT and TUNEL in the peri-infarct area on postoperative day 3 (n = 5). Scale bar, 20 μm. (I) Representative Prussian blue staining images and quantitative analysis in the infarct border zone on postoperative day 3. (n = 5). Scale bar, 100 μm–20 μm. Data are presented as mean ± SD. P values were calculated by one-way ANOVA with Tukey’s post-hoc test

• 

  Fig. 6. PMS@ME inhibits cardiomyocyte apoptosis in vitro. (A) Relative viabilities of HL-1 cells in the PDA, MnSe, or PMS treatment groups (n = 3). (B) Flow cytometry analysis of HL-1 cardiomyocytes stained with Annexin V-FITC and PI in the Control, H2O2, H2O2 + PDA, H2O2 + MnSe, and H2O2 + PMS groups (n = 3). (C) Representative Calcein AM/PI staining images of HL-1 cells in different groups (n = 3). Scale bar, 100 μm. (D) Relative viabilities of HL-1 cells after H2O2 injury and being treated with different concentrations of PMS for 4 h (n = 3). (E) Flow cytometry analysis of HL-1 cells stained with Annexin V-FITC and PI after being treated by different concentrations of PMS. Data are presented as mean ± SD. P values were calculated by one-way ANOVA with Tukey’s post-hoc test. (F) Representative Calcein AM/PI staining images of HL-1 cells after treatments with different concentrations of PMS for 4 h. Scale bar, 100 μm

• 

  Fig. 7. PMS@ME promotes post-MI cardiomyocyte proliferation and inhibits the Hippo signaling pathway. (A) Volcano plot showing significantly upregulated and downregulated genes in the comparison of the PMS@ME and H2O2 groups (n = 3). (B) Heat map of all differentially expressed genes in the Control, H2O2, and PMS@ME treatment groups. (C) Gene Ontology analysis of the most significantly regulated biological process (BP), cellular component (CC), molecular function (MF) in the PMS@ME versus H2O2 groups. (D) KEGG enrichment showing the upregulated and downregulated signaling pathways or biological processes in the PMS@ME versus H2O2 groups. (E) Gene Set Enrichment Analysis (GSEA) showing that DNA replication was significantly upregulated after PMS@ME treatment. (F) Representative immunofluorescence staining images of cTnT and Aurora B expression in each group on postoperative day 3 (n = 5). Scale bar, 20 μm. (G) Representative immunofluorescence images of cTnT and Ki67 expression in the infarct border zone on postoperative day 3 (n = 5). Scale bar, 50 μm–20 μm. (H) Representative immunofluorescence images of cTnT and phosphorylated H3 (pH3) expression in the infarct border zone on day 14…

• 

  Fig. 8. PMS@ME increases reparative immune cells and reduces pro-inflammatory immune cells and inflammatory responses. (A-D) Representative immunofluorescence/histochemistry images and quantitative analysis of F4/80+CD86+ macrophages, CD206+ macrophages, and MPO+ neutrophils in the peri-infarct area on postoperative day 3 (n = 5). Scale bar, 100–20 μm in F4/80 and CD86 staining, 20 μm in CD206 staining, and 100 μm in MPO staining. (E-G) Immunostaining and quantitative analysis of CD4 and Foxp3 expression in the infarct border zone on postoperative day 3 (n = 5). Scale bar, 20 μm. (H-K) Western blot analysis of IL-6, IL-1β, TNF-α, iNOS, Arg1, TGF-β, CD206, IL-10, HIF-1α, P-p38MAPK, TLR4, P65, and P-P65 in different groups on postoperative day 3 (n = 5). (L, M) Representative immunofluorescence images and quantitative analysis of iNOS expression in RAW264.7 cells from different groups (n = 3). Scale bar, 10 μm. Data are presented as mean ± SD for B-D, F, G, I-K, and M. P values were determined using one-way ANOVA followed by Tukey’s post-hoc test

• 

  Fig. 9. PMS@ME improves mitochondrial-related oxidative and metabolic homeostasis in cardiomyocytes. (A, B) DCFH-DA fluorescence staining and flow cytometry analysis of H2O2-stimulated HL-1 cells after treatments with PDA, MnSe and PMS. Scale bar, 100 μm. (C, D) Representative MitoSOX staining images and quantitative analysis of HL-1 cells in different groups. Scale bar, 20 μm. (E, F) Immunostaining analysis and flow cytometry of HL-1 cells stained with JC-1 (JC-1 polymer: red; JC-1 monomer: green) in the Control, H2O2, PDA, MnSe, and PMS groups (n = 3). Scale bar, 100 μm. (G) Oxygen Consumption Rate (OCR) and the calculated values of basal respiration, maximal oxygen consumption, ATP production in HL-1 cells after different treatments (n = 3). (H) ATP levels by absorbance utilizing an ATP assay kit in different groups. (I, J) DHE immunostaining and quantitative analysis for ROS in the peri-infarct area on postoperative day 3 (n = 5). Scale bar, 100 μm. (K) Western blot of PGC-1α and COX4 in different groups on postoperative day 3. Data are expressed as mean ± SD for B, D, F, G, and J. P values were calculated by one-way ANOVA with Tukey’s post-hoc test