Fibroblasts Rendered Antifibrotic, Antiapoptotic, and Angiogenic by Priming With Cardiosphere-Derived Extracellular Membrane Vesicles

Abstract

Background: Cardiosphere-derived cells mediate therapeutic regeneration in patients after myocardial infarction and are undergoing further clinical testing for cardiomyopathy. The beneficial effects of cardiosphere-derived cells are mediated by the secretion of exosomes and possibly other extracellular membrane vesicles (EMVs).

Objectives: This study sought to investigate the effect of cardiosphere-derived EMVs (CSp-EMVs) on fibroblasts in vitro and tested whether priming with CSp-EMVs could confer salutary properties on fibroblasts in vivo.

Methods: CSp-EMVs were isolated from serum-free media conditioned for 3 days by cardiospheres. Dermal fibroblasts were primed with CSp-EMVs for 24 h followed by exosomal micro-ribonucleic acid profiling. In vivo, we injected CSp-EMV-primed or -unprimed dermal fibroblasts (or CSp-EMVs) in a chronic rat model of myocardial infarction and defined the functional and structural consequences.

Results: CSp-EMVs amplified their own biological signals: exposure of "inert" fibroblasts to CSp-EMVs rendered the fibroblasts therapeutic. Intramyocardially injected CSp-EMV-primed (but not unprimed) fibroblasts increased global pump function and vessel density while reducing scar mass. CSp-EMV priming caused fibroblasts to secrete much higher levels of stromal-cell-derived factor 1 and vascular endothelial growth factor and dramatically changed the micro-ribonucleic acid profile of fibroblast-secreted EMVs in vitro. The priming was followed by significant angiogenic and cardioprotective effects.

Conclusions: CSp-EMVs alter fibroblast phenotype and secretome in a salutary positive-feedback loop. The phenotypic conversion of inert cells to therapeutically active cells reveals a novel mechanism for amplification of exosome bioactivity.

Keywords: cardiac repair; conversion; exosome; growth factor.

CENTRAL ILLUSTRATION. Conversion of Inert Fibroblasts to Active Cells

Cardiosphere-isolated exosomes were used to prime inert fibroblasts. Post-priming analysis of fibroblast bioactivity revealed amplification of their therapeutic properties including cardiomyogenic, angiogenic, antifibrotic, and regenerative effects.

FIGURE 1. CSp-EMV Characterization and Internalization

(A) Extracellular membrane vesicles (EMV) were isolated from cardiospheres (CSp) on day 3 post-plating by adding Exoquick precipitation solution. (B) Size distribution was analyzed by Nanoparticle Tracking Analysis and pooled data for particle number and size quantification revealed an average size of 175 ±12 nm diameter vesicles. (C) Tetraspanin-bound beads were used to characterize the human CSp-derived EMVs (hCSp-EMV). Representative histograms revealed expression of CD63, CD81, and CD9. EMVs stained for tetraspanins (green line) were compared to appropriate controls (orange/blue lines). (D) hDFs were incubated with fluorescent dyed hCSp-EMV for 24 h followed by confocal imaging. (E) z-stack image of DFs 24-h post-hCSp-EMV incubation revealed particle internalization. (F) Representative confocal images of DFs incubated with different concentrations of hCSp-EMV and evaluation of fluorescent intensity at different time points post-incubation revealed cells with EMV signal and EMV intensity per cell at 6 h (G and H), 12 h (I and J), and 24 h (K and L) that were dose but not time dependent. Scale bar = 250 μm; n = 3-5 high-power (20×) images per group. DAPI = 4',6-diamidino-2-phenylindole; DF = dermal fibroblast; WGA = wheat germ agglutinin.

FIGURE 2. In Vitro Characterization of CSp-EMV Biological Activity

Western blot of hDFs 24 h post-incubation with 2 different concentrations of hCSp-EMV showed reduced psmad2/3 (A), psmad4 (B), and snai1 (C). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a control. The experiment was performed in triplicates. Flow cytometry was used for phenotypic characterization of the hCSp–EMV-primed fibroblasts 24 h post-incubation. Representative FACS plots (D) and pooled data (E) revealed reduced fibroblast-specific protein 1 (FSP1) and discoidin domain receptor 2 (DDR2) expression while CD105 and CD90 were not affected (n = 3 in each group). Confocal images of hCSp-EMV-primed and -unprimed fibroblasts (F) showed enhanced density of the smooth muscle actin (SMA) cells and lower FSP1 density post-CSp-EMV incubation (G). Scale bar = 250 μm. (H) Scheme of the collected supernatant post hCSp-EMV treated fibroblasts; (I) enzyme-linked immunosorbent assay (ELISA) on the collected supernatant revealed increased levels of SDF-1 and VEGF in the post primed fibroblasts. n = 5 per group. *p < 0.05 vs. unprimed fibroblasts. FACS = fluorescence activated cell sorting; ISO = isotype control; smad = small mothers against decapentaplegic homolog; other abbreviations as in Figure 1.

FIGURE 3. In Vitro Bioactivity of CSp-EMV-primed Fibroblasts

Representative FACS plots of neonatal rat ventricular myocytes (NRVM) were treated with (A) hDF-EMVs, (B) conditioned media from hCSp-EMV-primed hDFs, and (C) hCSp-EMVs for 72 h and stained with Annexin V to evaluate apoptosis. The experiment was performed in triplicates (n = 3 for each group). (D) Pooled data for the NRVM apoptosis revealed higher viability in the hCSp-EMV-primed fibroblast and hCSp-EMV groups compared to the DF-EMVs. (Histogram color = group in bar graph.) E-G. Representative higher power images of the Matrigel tube formation assay from (E) hDF-EMVs, (F) conditioned media from hCSp-EMV-primed hDFs, and (G) hCSp-EMVs and (H) pooled data for tube quantification. Similarly, enhanced tube formation was observed in the latter 2 groups compared to the hDF-EMV only. *p < 0.05 vs. DF. Scale bar = 50 μm. Abbreviations as in Figures 1 and 2.

FIGURE 4. MiRNA Array Analysis: Signature of Secreted Extracellular Vesicles and Parent Cells

Micro-ribonucleic acid (miRNA) with statistically significant fold changes are seen in (A) hCSps versus hDFs and (B) hCSp-EMV-primed DFs versus unprimed hDFs. Additionally, fold ratios of miRNA profiles from EMVs derived either from hCSps or unprimed hDFs (C) and fold changes in the miRNA cargo of the EMVs secreted by hCSp-EMV-primed hDFs versus unprimed hDFs (D) are seen. All p<0.05. n = 3 per group. Abbreviations as in Figure 1.

FIGURE 5. In Vivo Studies

According to the study timeline (A), myocardial infarction was induced in Wistar Kyoto rats; 1 month later, the animals were allocated to injection of vehicle (PBS; orange bars; n = 6), rDFs (yellow; n = 8), rDFs primed with rCSp-EMV (green; n = 8), or rCSp-EMV only (blue; n = 8). Functional follow-up and histological analysis were performed 1 month post-injection. At 1 month post-injection, rCSp-EMV and rCSp-EMV-primed rDFs demonstrated significant improvement in cardiac function via ejection fraction (B) as well as better-maintained left ventricular end-systolic diameter (LVESD) via M-mode short axis images (C) compared to control groups. Scar mass was evaluated by serial Masson's Trichrome stained sections from the left ventricle (D), and was significantly reduced in the rCSp-EMV and the rCSp-EMV-primed rDF groups compared to either control group (E).Significant differences also were observed in infarct wall thickness (F). *p<0.05 vs. DFs; **p < 0.05 vs. PBS. PBS = phosphate-buffered saline; rCSp = rat cardiosphere; rDFs = rat dermal fibroblasts; other abbreviations as in Figure 1.

FIGURE 6. In Vivo Vessel Density 1-month Post-treatment

(A) Representative immunostained images from the infarct, border, and remote zones are presented for evaluation of microvessel and capillary density. In the infarct (B), border (C), and remote (D) zones, pooled data revealed enhanced von Willebrand factor (vWf)-positive capillary density in the rCSp-EMV and rCSp-EMV-primed rDF groups compared to both controls (left panels) and changes regarding SMA-positive vessels (right panels). n = 5 in each of the groups. Scale bar = 250 μm. *p < 0.05 vs. DFs; **p < 0.05 vs. PBS. Abbreviations as in Figures 1, 2, and 5.

FIGURE 7. In Vivo Cardiomyocyte Architecture 1-month Post-therapy

(A) Representative immunostained images from the border and remote zones were used for evaluation of cardiomyocyte diameter. Pooled data revealed no difference between the groups analyzed in the border (B) and remote (C) zones. n = 5 in each of the groups. Scale bar = 100 μm. ASA =α- sarcomeric actin; other abbreviations as in Figures 1 and 5.