Abstract 12532: Extracellular Vesicles Released by Cardiosphere-Derived Cells Dampen the Pro-Inflammatory Monocyte Response Through Repression of C-C Chemokine Receptor Type 2 (CCR2)

Abstract

Background: Following a myocardial infarction (MI), CCR2+ monocytes migrate toward the injury site along a CCL2 concentration gradient and play an integral role in removing debris and facilitating repair. Although required to orchestrate the pro-inflammatory response, prolonged activation of CCR2+ monocytes can lead to adverse outcomes. Extracellular vesicles (EVs) derived from cardiosphere-derived cells (CDCs) have been suggested to mediate cardioprotection in part via immunomodulation. We hypothesized that CDC-EVs influence CCR2+ monocyte recruitment and function. Methods: Blood samples were obtained from healthy human donors under approved institutional IRBs. Human monocytes were isolated from peripheral blood using density gradient centrifugation (Ficoll) followed by magnetic activated cell sorting (MACS). EVs were isolated from CDCs, normal human dermal fibroblasts (NHDFs, an inert population in models of ischemia), and CDCs transfected with a miR-146a antagomir. EVs were characterized according to established guidelines for size, morphology and protein expression. Monocytes were incubated with PBS, NHDF-EVs, CDC-EVs, or miR-146a deficient CDC-EVs and analyzed for CCR2 protein expression by flow cytometry and western blot and migration along a CCL2/MCP-1 gradient by in vitro chemotaxis assays. Results: CDC-EVs significantly reduced CD14 hi CD16 - monocyte surface expression of CCR2 by 50% as quantified by flow cytometry when compared with PBS and NHDF-EVs (p<0.05, one-way ANOVA, n=4). This appeared to be mediated by miR-146a as monocytes treated with CDC-EVs deficient in miR-146a had no significant difference in CCR2 levels compared with cells treated with PBS and NHDF-EV controls. Importantly, reduced levels of monocyte CCR2 expression following CDC-EV treatment led to a greater than 35% functional reduction in monocyte migration towards CCL2 compared to PBS control (42.8 ± 3.2% vs. 68.3 ± 4.9%, p<0.05, t-test, n=4). CCR2 expression remained suppressed for 8-12 hours and could be maintained with repeated doses. Conclusions: CDC-EVs modulate classical monocyte expression of CCR2 and migration towards CCL2/MCP-1 via miR-146a, elucidating a novel mechanism of CDC-EV immunomodulation.