Combinatorial treatment of acute myocardial infarction using stem cells and their derived exosomes resulted in improved heart performance

Abstract

Background: Bone marrow mesenchymal stem cells (MSCs) are among the most common cell types to be used and studied for cardiac regeneration. Low survival rate and difficult retention of delivered MSCs in infarcted heart remain as major challenges in the field. Co-delivery of stem cell-derived exosomes (Exo) is expected to improve the recruitment and survival of transplanted MSCs.

Methods: Exo was isolated from MSCs and delivered to an acute myocardial infarction (AMI) rat heart through intramyocardial injection with or without intravenous infusion of atrovastatin-pretreated MSCs on day 1, day 3, or day 7 after infarction. Echocardiography was performed to evaluate cardiac function. Histological analysis and ELISA test were performed to assess angiogenesis, SDF-1, and inflammatory factor expression in the infarct border zone. The anti-apoptosis effect of Exo on MSCs was evaluated using flow cytometry and Hoechst 33342 staining assay.

Results: We found that intramyocardial delivery of Exo followed by MSC transplantation (in brief, Exo+MSC treatment) into MI hearts further improved cardiac function, reduced infarct size, and increased neovascularization when compared to controls treated with Exo or MSCs alone. Of note, comparing the three co-transplanting groups, intramyocardially injecting Exo 30 min after AMI combined with MSCs transplantation at day 3 after AMI achieved the highest improvement in heart function. The observed enhanced heart function is likely due to an improved microenvironment via Exo injection, which is exemplified as reduced inflammatory responses and better MSC recruitment and retention. Furthermore, we demonstrated that pre-transplantation injection of Exo enhanced survival of MSCs and reduced their apoptosis both in vitro and in vivo.

Conclusions: Combinatorial delivery of exosomes and stem cells in a sequential manner effectively reduces scar size and restores heart function after AMI. This approach may represent as an alternative promising strategy for stem cell-based heart repair and therapy.

Keywords: Exosomes; MSCs; Myocardial infarction; SDF-1.

Fig. 1

Characterization and functional validation of exosomes derived from MSCs (Exo). a Phenotypic analysis of cell surface antigens of MSCs by flow cytometry. b Cup-shaped morphology of purified Exo assessed by transmission electron microscopy. Scale bar = 200 nm. c The particle size, particle concentration, and video frame of Exo analyzed by nanoparticle tracking analysis. d Representative images of Western blot showing the exosomal protein markers. e Representative confocal images showing that red fluorescence dye PKH26-labeled Exo (Exo-PKH26) were endocytosed by MSCs after12-h incubation. Scale bar = 60 μm. f Distribution of Exo-PKH26 in the infarcted heart on day 1, day 3, and day 7 post injection. Scale bar = 50 μm. g Quantification of PKH26+ cells in f (n = 5). (A-G) n = 5. All data are mean ± SEM. Statistical analysis was performed with one-way ANOVA followed by Tukey’s test. **p < 0.01, ****p < 0.0001

Fig. 2

Exo and MSCs synergistically improved cardiac function and ameliorated fibrosis after AMI. a Schematic of the sequential delivering of Exo followed by MSCs into the infarcted hearts experiment. b Representative echocardiogram of rat hearts in different groups 5 weeks post AMI. c, f Significantly enhanced left ventricular ejection fraction (LVEF), fractional shortening (LVFS), left ventricular end-diastolic volume (LVEDV), and left ventricular end-systolic volume (LVESV) in rats co-transplanted with Exo and MSCs compared with AMI, Exo, and MSCs alone groups (n = 6–8 for each group). g Representative transverse heart sections analyzed with Masson trichrome staining at 5 weeks after AMI. Red, myocardium; blue, scarred fibrosis. Scale bar = 2 mm. h Representative Sirius Red staining images for collagen analyze in each group. Scale bar = 2 mm. i, j Quantitative data for the LV fibrotic area and the quantitative data of Masson trichrome staining (i) and Sirius Red staining (j) (n = 6–8 for each group). All data are mean ± SEM. Statistical analysis was performed with two-way ANOVA followed by Tukey post hoc test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. AMI group; ^#p < 0.05, ^##p < 0.01, ^###p < 0.01 vs. Exo group; ^$p < 0.05, ^$$p < 0.01, ^$$$$p < 0.0001 vs. MSC group; ^&p < 0.05 vs. Exo + MSC^d7 group

Fig. 3

Exo and MSCs combined therapy promoted angiogenesis in the infarcted hearts. a Arterioles at the border zone on 5 weeks after AMI were identified by staining with α-SMA (green) and nuclei (blue). b Quantification of α-SMA+ cells in A (n = 5). c Capillary at the border zone on 5 weeks after AMI was identified by staining with CD31 (green) and nuclei (blue). d Quantification of CD31+ cells in c (n = 5). All data are mean ± SEM. Statistical analysis was performed with two-way ANOVA followed by Tukey post hoc test. *p < 0.05, **p < 0.01, ****p < 0.0001 vs. AMI group; ^##p < 0.01, ^####p < 0.0001 vs. Exo group; ^$$p < 0.01, ^$$$p < 0.001, ^$$$$p < 0.0001 vs. MSC group; ^&&p < 0.01, ^&&&&p < 0.0001 vs. Exo + MSC^d1 and Exo + MSC^d7 groups. Scale bar = 50 μm

Fig. 4

Exo pretreatment provided a relatively low inflammatory reaction and relatively high level of SDF-1 micro-environment in infarcted hearts for transplanted MSCs to survive. a Distribution of MSCs pre-labeled with CM-Dil (red) in the infarcted hearts on 5 weeks post AMI in the groups with and without PKH67 pre-labeled Exo (green) injection. Scale bar = 100 μm. b Quantification of CM-Dil+ cells in A (n = 5). c Representative HE staining images at the border zone 5 weeks after AMI. Scale bar = 200 μm. d, e Quantification of IL-6 and TNF-α expression level in the infarct border zone tissue of rat hearts using ELISA method (n = 5). f Representative immunohistochemical staining images of SDF-1 in PBS and Exo groups in 1 day, 3 days, and 7 days post AMI (× 200). Scale bar = 200 μm. g ELISA assessment of SDF-1 expression during the time course. All data are mean ± SEM. Statistical analysis was performed with one-way ANOVA followed by Tukey’s test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ^##p < 0.01, ^###p < 0.001

Fig. 5

Exo enhanced the survival and decreased the apoptosis of MSCs under hypoxia and serum deprivation (H/SD) conditions. a–d Flow cytometry assay and Hoechst 33342 nucleic acid stain for apoptosis of MSCs after treated with PBS, Exo, or Exo (Ultra) and exposed to H/SD. a Scatter diagram of apoptosis in MSCs treated with Exo and negative control. b Histogram of apoptosis events in different groups (n = 3). c Representative images of Hoechst 33342 nucleic acid stain. Scale bar = 100 μm. d Quantification of apoptosis cells in each group (n = 3). e Western blot for Bcl-2 protein in MSCs treated with Exo or Exo (Ultra) and exposed to H/SD condition. f Quantification of Bcl-2 in E (n = 3). g TUNEL staining at the border zone on 5 weeks post AMI with TUNEL (green) and nuclei (blue). h Quantification of TUNEL+ cells in G (n = 5). Apoptosis rate was quantified as the percentage of cells that were positive for TUNEL staining. All data are mean ± SEM. Statistical analysis was performed with one-way ANOVA followed by Tukey’s test or two-way ANOVA followed by Tukey post hoc test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. H/SD or AMI group; ^###p < 0.001 vs. Exo group; ^$p < 0.05, ^$$p < 0.01 vs. H/SD + Exo or MSC group; ^&p < 0.05 vs. Exo + MSC^d1 group. Scale bar = 50 μm

Fig. 6

Effects and mechanisms of MSC-derived exosomes and MSC combinatorial therapy on acute myocardial infarction. Delivery of Exo into infarcted hearts 30 min post AMI can significantly reduce inflammatory factors such as IL-6 and TNF-α, increase SDF-1 expression and angiogenesis, and promote cell survival in the stressed ischemic microenvironment at day 3 post AMI. These mechanisms ultimately lead to significant augmentation of cardiac function and reducing scar size of AMI rat hearts after Exo and stem cells combinatorial therapy