Introduction: Cardiosphere-derived cells (CDCs), which are in advanced clinical testing for heart failure, Duchenne muscular dystrophy and pulmonary hypertension, secrete exosomes (CDCexo) which are highly anti-inflammatory. CDCexo, but not fibroblast (FB) exosomes, lead to cardioprotection through polarization of macrophages (Mφ) following ischemia/reperfusion injury (de Couto et al., Circulation, 2017). Here we demonstrate that intravenous delivery of CDCexo in mice (healthy or acutely inflamed) leads to preferential uptake by resident Mφ via phagocytosis.
Methods & Results: To assess biodistribution, we labeled CDCexo with the fluorescent liposomal dye DiD (Invitrogen). Healthy mice (C57Bl6; 8-12 weeks) were treated with an intraperitoneal injection of either LPS (5mg/kg) or saline prior to intravenous infusion of DiD-labeled CDCexo (in 100μL saline). Four hours later, organs were collected for ex vivo analysis. Biofluorescence imaging (Xenogen) revealed strong DiD signal in Mφ-rich organs including the liver, spleen, and lung, while immunohistochemistry and flow cytometry resolved preferential uptake of CDCexo by tissue Mφ within the liver (60-80% DiD+CD11b+), spleen (70-80% DiD+F4/80+), lung (60-90% DiD+F4/80+), and peritoneal cavity (40-60% DiD+CD11b+). The heart also exhibited measurable fluorescence signals. To determine how avidly distinct cardiovascular cell types preferentially take up CDCexoin vitro, Mφ, FB, endothelial cells (EC), and cardiomyocytes (CM) were exposed to DiD-labeled CDCexo and examined over time. Within one hour, Mφ demonstrated the greatest uptake of CDCexo (Mφ: 81%; FB: 60%; EC: 65%; CM: 51%; p<0.05). The addition of both phagocytosis (Cytochalasin D) and clathrin (Pitstop 2) inhibitors completely blocked CDCexo uptake by Mφ.
Conclusions: Intravenous delivery of CDCexo in mice (healthy or acutely inflamed) leads to preferential uptake by Mφ within organs that are rich in such cells (i.e., liver, spleen, lung, and peritoneal cavity). In vitro, Mφ take up CDCexo via phagocytosis and endocytosis more readily than other cardiac cell types. These data suggest the potential utility of CDCexo treatment in cardiovascular diseases where Mφ infiltration is prominent (e.g., atherosclerosis).