Identification of Myeloid Protein Kinase C Epsilon as a Novel Atheroprotective Gene.

BACKGROUND: Atherosclerosis is a chronic inflammatory disease driven by macrophages. PKCɛ (protein kinase C epsilon) is a serine/threonine kinase involved in diverse cellular processes including migration, growth, differentiation, and survival. PKCɛ acts in a context-dependent manner within the heart; however, its role in atherosclerosis is unknown. METHODS: Bone marrow-derived macrophages from global PKCɛ knockout mice were tested for lipid retention and cytokine secretion. Public gene set analysis assessed raw counts of PRKCE in human atheromas to determine translational relevance. A LysM Cre PKCɛ(fl/fl) (myeloid-selective PKCɛ knockout [mɛKO]) mouse was developed to study the impact of myeloid PKCɛ on atherosclerosis. After confirming myeloid-selective PKCɛ deletion, human-like hypercholesterolemia was induced, and multiple metrics of atherosclerosis were compared between wild-type (WT) and mɛKO plaques. RNA sequencing was used to provide unbiased insight into possible mechanisms by which PKCɛ regulates atherosclerosis. RESULTS: Public gene set analysis of human atherosclerotic plaque tissue revealed that PRKCE expression is inversely correlated with plaque vulnerability. Similarly, peritoneal macrophages from WT hypercholesterolemic mice have significantly lower PKCɛ expression, providing a translational rationale for the generation of the mɛKO mouse. Quantitative polymerase chain reaction revealed no differences between genotypes in the expression of genes related to atherosclerosis, at either steady state or upon lipid loading, suggesting that loss of PKCɛ does not fundamentally change the basal state and that differences seen are a result of a more complex pathway. Comparing descending aorta and aortic root plaques from WT and mɛKO hypercholesterolemic mice revealed that mɛKO plaques are larger, have larger foam cells and regions of necrosis, and thinner collagen caps. Upon lipid loading in vitro and in vivo, mɛKO macrophages retained significantly more cholesterol and lipid droplets than WT; Gene Ontology suggests higher expression of genes related to endocytosis in mɛKO macrophages compared with WT. CONCLUSIONS: PRKCE expression is decreased in vulnerable human plaques and PKCɛ decreases in mouse macrophages upon lipid loading. mɛKO plaques are larger and exhibit markers of vulnerability. With no differences in SR (scavenger receptor) expression, the impact of PKCɛ deletion is more subtle than simple SR dysregulation. RNA sequencing implicates higher expression of genes involved in endocytosis, and mɛKO macrophages have significantly more lipid-containing endosomes. The data define the atherophenotype of mɛKO mice and demonstrate that PKCɛ restricts lipid uptake into macrophages by a mechanism independent of SR expression. Taken together, these studies identify PKCɛ as a novel atheroprotective gene, laying the foundation for mechanistic studies on the endocytic signaling networks responsible for the phenotype.