Experimental, Systems, and Computational Approaches to Understanding the MicroRNA-Mediated Reparative Potential of Cardiac Progenitor Cell-Derived Exosomes From Pediatric Patients

Abstract

Rationale: Studies have demonstrated that exosomes can repair cardiac tissue post-myocardial infarction and recapitulate the benefits of cellular therapy.

Objective: We evaluated the role of donor age and hypoxia of human pediatric cardiac progenitor cell (CPC)-derived exosomes in a rat model of ischemia-reperfusion injury.

Methods and results: Human CPCs from the right atrial appendages from children of different ages undergoing cardiac surgery for congenital heart defects were isolated and cultured under hypoxic or normoxic conditions. Exosomes were isolated from the culture-conditioned media and delivered to athymic rats after ischemia-reperfusion injury. Echocardiography at day 3 post-myocardial infarction suggested statistically improved function in neonatal hypoxic and neonatal normoxic groups compared with saline-treated controls. At 28 days post-myocardial infarction, exosomes derived from neonatal normoxia, neonatal hypoxia, infant hypoxia, and child hypoxia significantly improved cardiac function compared with those from saline-treated controls. Staining showed decreased fibrosis and improved angiogenesis in hypoxic groups compared with controls. Finally, using sequencing data, a computational model was generated to link microRNA levels to specific outcomes.

Conclusions: CPC exosomes derived from neonates improved cardiac function independent of culture oxygen levels, whereas CPC exosomes from older children were not reparative unless subjected to hypoxic conditions. Cardiac functional improvements were associated with increased angiogenesis, reduced fibrosis, and improved hypertrophy, resulting in improved cardiac function; however, mechanisms for normoxic neonatal CPC exosomes improved function independent of those mechanisms. This is the first study of its kind demonstrating that donor age and oxygen content in the microenvironment significantly alter the efficacy of human CPC-derived exosomes.

Keywords: cardiac progenitor cells; exosome; microRNA; modeling; systems biology.

Figure 1. Characterization and exosomal uptake

A) Isolated exosomes from human pediatric CPCs were identified via Transmission Electron Microscopy. B–D) Uptake of Exosomes - Exosomes were taken up by the (B) Rat cardiac myocytes, (C) Rat endothelial cells and (D) rat cardiac fibroblast cells. Exosomes were labeled with Acridine Orange (green) and nucleus was stained with Hoechst (blue) dye. Z-stacks are given below and to the right of the confocal image.

Figure 2. Functional Analysis via 2D echo

Graph represents mean ± SEM of Ejection Fraction (%) at day 3 (A) and day 28 (B) days post MI, (n=4–5), Blue = Normoxia, Red = Hypoxia. *p <0.05; ANOVA followed by Tukey-Kramer post-test in comparison to the saline-treated control animals.

Figure 3. Fibrosis analysis

Picrosirius staining of paraffin embedded infarcted hearts. A–E) Representative images as labeled. F) Grouped data represents mean ± SEM of % LV fibrosis. Hypoxic exosomes from each age group (neonate, infant and child (n=4)) showed significantly decreased fibrosis compared with controls (n=5) and their normoxic counterparts (neonate (n=4), infant (n=5) and child (n=5)). *p<0.05; ANOVA followed by Tukey-Kramer post-test.

Figure 4. Angiogenesis Analysis

Immunostaining for Isolectin (Green) and capillary quantification was performed. A–D) Representative images as labeled. Capillaries were quantified by counting total number of capillaries per unit area in 3 cross-sectional areas from the infarcted region of LV of each animal. E). Grouped data represents mean ± SEM of capillaries/mm². Hypoxic exosomes from each age group (neonate, infant and child(n=4)) showed significantly increased angiogenesis compared with saline-treated controls (n=5) and their normoxic counterparts (neonate (n=4), infant (n=5) and child (n=5)). *p<0.05; ANOVA followed by Tukey-Kramer post-test.

Figure 5. Hypertrophy Analysis

Hypertrophy was analyzed in the peri-infarct region by immunostaining and left ventricular posterior wall diameter (LVPWd) by 2D echo. A–D) Immunostaining for wheat germ agglutinin (Red) and nuclei (DAPI) was performed for peri-infarct hypertrophy. Cross-sectional area of cardiac myocytes at the level of the nuclei in-plane in the peri-infarct region was determined at 3 different areas of each animal. A–C) Representative images as labeled. D). Grouped data represents mean ± SEM of cross section area of a myocyte (µm²). Exosomes from all groups showed significantly decreased hypertrophy compared with control (n=4–5). *p<0.05; ANOVA followed by Tukey-Kramer post-test. E) M-mode images of 2D echo were quantified for determining LVPWd. Grouped data represents mean ± SEM of LVPWd. Exosomes from neonatal hypoxia showed significant decreased hypertrophy compared with controls and neonatal normoxia groups. Exosomes from infant hypoxia showed significant decreased hypertrophy compared with controls. *p<0.05; Unpaired t-test.

Figure 6. Computational model of exosome study

A) Principal Component (PC) Analysis. All age groups cluster in unique components in the first PC analysis. Hypoxia is predicted to have largest effect. B) PLSR Analysis. Top microRNAs with known targets were identified by PLSR and plotted in PC space. Figure represents miR clustering in based upon the outcomes.

Figure 7. Integrated model with pediatric CPC and CD34+ cell-derived exosomes

A) Predictive model was created using array data and functional data from this study and ref 20 using top 30 microRNAs from Figure 6. B) Model results were closely aligned with published studies from (20). C) PC analysis of CPC, CD34+ cell, and mononuclear cells (MNCs) showed tight clustering of CD34+ cell-derived exosomes and newborn CPC exosomes. D) PLSR analysis identified potential miRs from both studies that contribute toward function.

Figure 8. Infarct size in rats treated with newborn exosomes

Top panel shows graph represented as infarct size/area at risk (AAR) in rats treated with saline (IR), or exosomes derived from newborn CPCs cultured under normoxia or hypoxia. Bottom panel shows representative images from the 3 groups. Data are mean ± SEM (n=4 per group). *p<0.05 ANOVA followed by Tukey-Kramer post-test.