The Intrapericardial Delivery of Extracellular Vesicles from Cardiosphere-Derived Cells Stimulates M2 Polarization during the Acute Phase of Porcine Myocardial Infarction

Abstract

Acute myocardial infarction triggers a strong inflammatory response in the affected cardiac tissue. New therapeutic tools based on stem cell therapy may modulate the unbalanced inflammation in the damaged cardiac tissue, contributing to the resolution of this pathological condition. The main goal of this study was to analyze the immunomodulatory effects of cardiosphere-derived cells (CDCs) and their extracellular vesicles (EV-CDCs), delivered by intrapericardial administration in a clinically relevant animal model, during the initial pro-inflammatory phase of an induced myocardial infarction. This effect was assessed in peripheral blood and pericardial fluid leukocytes from infarcted animals. Additionally, cardiac functional parameters, troponin I, hematological and biochemical components were also analyzed to characterize myocardial infarction-induced changes, as well as the safety aspects of these procedures. Our preclinical study demonstrated a successful myocardial infarction induction in all animals, without any reported adverse effect related to the intrapericardial administration of CDCs or EV-CDCs. Significant changes were observed in biochemical and immunological parameters after myocardial infarction. The analysis of peripheral blood leukocytes revealed an increase of M2 monocytes in the EV-CDCs group, while no differences were reported in other lymphocyte subsets. Moreover, arginase-1 (M2-differentiation marker) was significantly increased in pericardial fluids 24 h after EV-CDCs administration. In summary, we demonstrate that, in our experimental conditions, intrapericardially administered EV-CDCs have an immunomodulatory effect on monocyte polarization, showing a beneficial effect for counteracting an unbalanced inflammatory reaction in the acute phase of myocardial infarction. These M2 monocytes have been defined as "pro-regenerative cells" with a pro-angiogenic and anti-inflammatory activity.

Keywords: Acute myocardial infarction; Cardiosphere-derived cells; Extracellular vesicles; Inflammation; Intrapericardial administration.

Fig. 1

Experimental design. At day 0, infarct model was created (white arrow). At 72 h, magnetic resonance imaging was performed (black arrow). Placebo, EV-CDCs or CDCs were intrapericardially administered at 72 h (grey arrow). Blood samples were collected at day 0 (Basal), 72 h (post-AMI) and 24 h after intrapericardial administration (post-therapy). Blood samples were used for flow cytometry analysis (triangles), hematology (squares) and biochemical analysis (rhombus)

Fig. 2

White blood cell analysis in peripheral blood. Blood samples were collected in EDTA containing tubes before acute myocardial infarction model creation (Basal), 72 h after (Post-AMI) and 24 h after the treatment (Post-therapy) and white blood cells were counted in an automated hematology analyzer. Normality was assessed using a Shapiro-Wilk test. Paired comparisons of the AMI model (a)(n = 15) and paired comparisons of the administered therapies (b) (n = 5) were performed using a Student t-test for parametric data or a Wilcoxon signed rank test with the Yates continuity correction for non-parametric variables. Graphs show the mean ± SD of cell populations. **p ≤ 0.01. ****p ≤ 0.0001

Fig. 3

Lymphocyte subsets distribution in peripheral blood. Peripheral blood lymphocytes were isolated from blood samples before acute myocardial infarction model creation (Basal), 72 h after (Post-AMI) and 24 h after the treatment (Post-therapy). Lymphocyte subsets distribution was analyzed by flow cytometry. Normality was assessed using a Shapiro-Wilk test. Paired comparisons of the AMI model (a)(n = 15) and paired comparisons of the administered therapies (b) (n = 5) were performed using a Student t-test for parametric data or a Wilcoxon signed rank test with the Yates continuity correction for non-parametric variables. Graphs show the mean ± SD of cell populations. *p ≤ 0.05. **p ≤ 0.01. ***p ≤ 0.001. ****p ≤ 0.0001

Fig. 4

Differentiation/activation T cell subsets status in peripheral blood. Peripheral blood lymphocytes were isolated from blood samples before acute myocardial infarction model creation (Basal), 72 h after (Post-AMI) and 24 h after the treatment (Post-therapy). T cell subset status was analyzed by flow cytometry using CD27 and CD45RA markers. The co-expression analysis of these two markers allowed us to identify naïve T cells (CD27+ CD45RA+) and effector/memory T cells (CD27- CD45RA-). Normality was assessed using a Shapiro-Wilk test. Paired comparisons of the AMI model (a) (n = 15) and paired comparisons of the administered therapies (b) (n = 5) were performed using a Student t-test for parametric data or a Wilcoxon signed rank test with the Yates continuity correction for non-parametric variables. Graphs show the mean ± SD of cell populations. *p ≤ 0.05. **p ≤ 0.01. ***p ≤ 0.001. ****p ≤ 0.0001

Fig. 5

Monocyte populations in peripheral blood. Blood samples were collected in EDTA containing tubes before acute myocardial infarction model creation (Basal), 72 h after (Post-AMI) and 24 h after the treatment (Post-therapy). Monocyte count was performed in an automated hematology analyzer and its phenotype characterization was evaluated by flow cytometry, defining circulating M2 monocytes as CD14 + CD163+. Normality was assessed using a Shapiro-Wilk test. Paired comparisons of the AMI model (a)(n = 15) and paired comparisons of the administered therapies (b) (n = 5) were performed using a Student t-test for parametric data or a Wilcoxon signed rank test with the Yates continuity correction for non-parametric variables. Graphs show the mean ± SD of cell populations. **p ≤ 0.01

Fig. 6

Cytokines gene expression in pericardial fluid cells. Pericardial fluids were compiled before and 24 h after EV-CDCs administration. Total RNA was isolated from pericardial fluid cells and qPCR products were quantified by the 2^-∆Ct method using GAPDH as an endogenous control. The statistical analysis was performed using a Thermo Fisher Cloud Analysis version 1.0. Graphs show the mean ± SD (n = 3). *p ≤ 0.05