Subendothelial Accumulation of Exosomes and Coronary Microvascular Dysfunction in Mice Lacking Acid Ceramidase

Abstract

Coronary microvascular dysfunction (CMD) has been demonstrated to be one of the major factors contributing to ischemic heart disease. However, the mechanisms initiating CMD remain poorly understood. The present study tested the hypothesis that lysosomal acid ceramidase finely controls subendothelial accumulation of exosomes leading CMD and subsequent myocardial ischemia. Using an endothelial-specific acid ceramidase (AC) gene (mouse gene code: Asah1) knockout (Asah1fl/fl/ECcre) mouse model fed with the Western diet (WD), we found that Asah1fl/fl/ECcre mice on the WD have more thickening of mid-layer in their small coronary arteries as shown significantly increased media/lumen ratio compared to the WD-fed wild type littermates (WT/WT). By nanoparticle tracking analysis (NTA) using NanoSight NS300 nanoparticle analyzer, we also observed that exosomes (~80-130 nm) in the blood stream were markedly increased in WD-fed Asah1fl/fl/ECcre mice compared to WT/WT mice. There were also more exosomes detected in the subendothelial space of coronary arterial wall, as shown by remarkable increase in colocalization of exosome marker CD63 or Annexin II with proteoglycans, bioglycan or decorin (subendothelial matrix markers) in Asah1fl/fl/ECcre mice. By electron microscopy (EM), we further confirmed that Asah1fl/fl/ECcre mice on the WD also significantly augmented the accumulation of exosomes under the endothelium small coronary arteries. Functionally, electrocardiogram (ECG) recording showed that in response to ergonovine, a coronary artery constrictor, the elevation of S-T segment was more remarkable in WD-fed Asah1fl/fl/ECcre than WT/WT mice. Using HypoxyProbe-1, the myocardial ischemic response to ergonovine was also detected more in the heart of WD-fed Asah1fl/fl/ECcre than WT/WT mice. Together, these results indicate that subendothelial exosome accumulation due to defect of lysosomal AC gene expression and activity may be a crucial pathogenic factor for CMD.