Extracellular Vesicles Promote Arteriogenesis in Chronically Ischemic Myocardium in the Setting of Metabolic Syndrome

Figures

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  Figure 1. Glucose tolerance test and body mass index (BMI) values. There was no significant difference in glucose tolerance between any of the groups at any of the time points. The 30‐ and 60‐minute values at all time points in all groups reflect impaired glucose tolerance consistent with metabolic syndrome. The BMI levels were consistently higher in the high‐fat diet with myocardial extracellular vesicle (EV) injection group (HVM) when compared with the control group at all time points; however, there was no significant difference in percentage change of weight gain between the groups. Data were analyzed using Student t test. CON indicates control group (high‐fat diet with vehicle injection); HVMC, HVM and calpain inhibition group. *P<0.05 was considered significant.

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  Figure 2. Blood flow is increased in the high‐fat diet with myocardial extracellular vesicle injection group (HVM) during pacing. Extracellular vesicle injection increased blood flow (mL/min per g) to both the ischemic and nonischemic areas of the left ventricle during paced conditions to 150 beats per minute in the HVM group. This was not recapitulated in the group that also received oral calpain inhibition (HVMC) nor was there any difference between groups when the heart was at rest. CON indicates control group (high‐fat diet with vehicle injection). *P<0.05 (Kruskal‐Wallis with Dunn's post hoc analysis test was used to compare the means among the 3 groups).

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  Figure 3. Cardiac output (CO) and stroke volume (SV), but not dP/dt, were increased in the high‐fat diet with myocardial extracellular vesicle (EV) injection group (HVM) during pacing. Pressure‐volume (PV) loop analyses were performed to determine cardiac function (top: a representative PV loop from each group). EV injection increased CO and SV during paced conditions to 150 beats per minute in the HVM group. This was not recapitulated in the group that also received oral calpain inhibition (HVMC) nor was there any difference between groups when the heart was at rest. dP/dt and stroke work (SW) are also shown. CON indicates control group (high‐fat diet with vehicle injection), LV, left ventricle. *P<0.05 (with linear regression used for analysis).

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  Figure 4. Extracellular vesicle (EV) injection improves arteriolar density in the myocardium of high‐fat fed animals. A, Arteriolar density, as determined by α‐smooth muscle actin (α‐SMA) staining, was significantly increased in the high‐fat diet with myocardial EV injection group (HVM) compared with the control group (high‐fat diet with vehicle injection) (CON) in both the nonischemic and the ischemic myocardium. There was no difference between the HVMC and calpain inhibition group (HVMC) and the CON in either the nonischemic or the ischemic ventricle. B, A ×20 high‐power field image representative of myocardial tissue with staining of α‐ in red, cluster of differentiation 31 (CD31) in green, and 4’,6‐diamidino‐2‐phenylindole (DAPI) in blue. *P<0.05 (Kruskal‐Wallis with Dunn's post hoc analysis test was used to compare the means among the 3 groups).

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  Figure 5. Calpain inhibition (CI) decreases apoptosis signaling in ischemic myocardium treated with extracellular vesicle (EV) injection in the setting of metabolic syndrome. There was no significant difference in expression of endothelial NO synthase (eNOS) or phosphorylated eNOS (p‐eNOS)/eNOS between either group compared with the control group (high‐fat diet with vehicle injection) (CON). The high‐fat diet with EV injection (HVM) and CI group (HVMC) had a significant decrease in p‐eNOS compared with CON, suggestive of inhibition of p‐eNOS activation by CI. B‐cell lymphoma‐2 (BCL‐2), BCL‐2–associated X protein (Bax), transforming growth factor‐β (TGF‐β), and vascular endothelial growth factor receptor 2 (VEGF‐R2) were not significantly different between groups. BCL‐2–associated death promoter (BAD) was significantly downregulated in the HVMC group compared with the control, suggesting a protective effect of CI against apoptosis. *P<0.05 (Kruskal‐Wallis with Dunn's post hoc analysis test was used to compare the means among the 3 groups).

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  Figure 6. Proteomic footprint of the extracellular vesicle (EV) proteome overlapped in 2 different sets of EV samples. A, The Venn diagram analysis of the unique proteins identified from each EV sample. The Venn diagram was generated using 2 publicly available bio tools (namely, Venny 2.1 [http://bioinfogp.cnb.csic.es/tools/venny/] and Pacific Northwest National Laboratory [http://omics.pnl.gov/software/venn-diagram-plotter]). B through E, The Gene Ontology (GO) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses of the overlapped proteins and the Online Mendelian Inheritance in Man (OMIM) analyses of the protein groups involved in disease/pathologies. GO and KEGG pathway analyses were done using Enrichr (http://amp.pharm.mssm.edu/Enrichr/), and OMIM analysis was performed using OMIM (https://omim.org/). F, Diseases that may result from dysfunction of the major proteins that are modulated in both groups of proteomics (taken from the common red area in the Venn diagram of A).