Identification of Myeloid Protein Kinase C Epsilon as a Novel Atheroprotective Gene
Background: Atherosclerosis is a chronic inflammatory disease driven by macrophages. PKC? (protein kinase C epsilon) is a serine/threonine kinase involved in diverse cellular processes including migration, growth, differentiation, and survival. PKC? acts in a context-dependent manner within the heart; however, its role in atherosclerosis is unknown. Methods: Bone marrow-derived macrophages from global PKC? 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 PKC?fl/fl (myeloid-selective PKC? knockout [m?KO]) mouse was developed to study the impact of myeloid PKC? on atherosclerosis. After confirming myeloid-selective PKC? deletion, human-like hypercholesterolemia was induced, and multiple metrics of atherosclerosis were compared in wild-type (WT) and m?KO plaques. RNA sequencing was used to provide unbiased insight into possible mechanisms by which PKC? regulates atherosclerosis. Results: Public gene set analysis of human atherosclerotic plaque tissue revealed that PKC? expression is inversely correlated with plaque vulnerability. Similarly, peritoneal macrophages from WT hypercholesterolemic mice have significantly lower PKC? expression, providing a translational rationale for the generation of the m?KO mouse. Quantitative polymerase chain reaction revealed no differences between genotypes in the expression of genes related to atherosclerosis, at either steady state or on lipid loading, suggesting that loss of PKC? 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 m?KO hypercholesterolemic mice revealed that m?KO plaques are larger, have larger foam cells and regions of necrosis, and thinner collagen caps. On lipid loading in vitro and in vivo, m?KO macrophages retained significantly more cholesterol and lipid droplets than WT; Gene Ontology suggests higher expression of genes related to endocytosis in m?KO macrophages compared with WT. Conclusions: PKC? expression is decreased in vulnerable human plaques and decreases in mouse macrophages on lipid loading. m?KO plaques are larger and exhibit markers of vulnerability. With no differences in SR (scavenger receptor) expression, the impact of PKC? deletion is more subtle than simple SR dysregulation. RNA sequencing implicates higher expression of genes involved in endocytosis, and m?KO macrophages have significantly more lipid-containing endosomes. The data define the atherophenotype of m?KO mice and demonstrate that PKC? restricts lipid uptake into macrophages by a mechanism independent of SR expression. Taken together, these studies identify PKC? as a novel atheroprotective gene, laying the foundation for mechanistic studies on the endocytic signaling networks responsible for the phenotype. Overall design: 8 week old mice (8 WT, 8 meKO) were injected with AAV8PCSK9 to remove LDL receptor from hepatocytes.  Combined with a high fat ("Western") diet, these animals become hypercholesterolemic.  4 WT and 4 meKO animals were fed the high fat diet; control animals (r of each genotype) remained on a chow diet.  4 weeks after AAV8/high fat diet, macrophages were recruited to the peritoneum with sterile thioglycolate and the elicited macrophages harvested by peritoneal lavage