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

Abstract

BACKGROUNDAtherosclerosis is a chronic inflammatory disease driven by macrophages (MO). Protein Kinase C - epsilon (PKC{varepsilon}) is a serine/threonine kinase involved in diverse cellular processes including migration, growth, differentiation, and survival. PKC{varepsilon} acts in a context dependent manner within the heart, however, its role in atherosclerosis is unknown. METHODSBone marrow derived MO from global PKC{varepsilon} knockout mice were tested for lipid retention and cytokine secretion. Public geneset analysis assessed raw counts of PRKCE in human atheromas to determine translational relevance. A LysM Cre PKC{varepsilon}fl/fl (m{varepsilon}KO) mouse was developed to study the impact of myeloid PKC{varepsilon} on atherosclerosis. After confirming myeloid selective PKC{varepsilon} deletion, human-like hypercholesterolemia was induced and multiple metrics of atherosclerosis were compared in WT and m{varepsilon}KO plaques. RNA sequencing was used to provide unbiased insight into possible mechanisms by which PKC{varepsilon} regulates atherosclerosis. RESULTSPublic geneset analysis of human atherosclerotic plaque tissue revealed that PKC{varepsilon} expression is inversely correlated with plaque vulnerability. Similarly, peritoneal MO from WT hypercholesterolemic mice have significantly lower PKC{varepsilon} expression, providing a translational rational for generation of the m{varepsilon}KO mouse. qPCR 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 PKC{varepsilon} 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{varepsilon}KO hypercholesterolemic mice revealed that m{varepsilon}KO plaques are larger, have larger foam cells and regions of necrosis, and thinner collagen caps. Upon lipid loading in vitro and in vivo, m{varepsilon}KO MO retained significantly more cholesterol and lipid droplets than WT; Gene Ontology suggests higher expression of genes related to endocytosis in m{varepsilon}KO MO compared to WT. CONCLUSIONSPKC{varepsilon} expression is decreased in vulnerable human plaques and decreases in mouse MO upon lipid loading. m{varepsilon}KO plaques are larger and exhibit markers of vulnerability. With no differences in scavenger receptor (SR) expression, the impact of PKC{varepsilon} deletion is more subtle than simple SR dysregulation. RNA sequencing implicates higher expression of genes involved in endocytosis and m{varepsilon}KO MO have significantly more lipid-containing endosomes. The data define the atherophenotype of m{varepsilon}KO mice and demonstrate that PKC{varepsilon} restricts lipid uptake into MO by a mechanism independent of SR expression. Taken together, these studies identify PKC{varepsilon} as a novel atheroprotective gene, laying the foundation for mechanistic studies on the endocytic signaling networks responsible for the phenotype. GRAPHIC ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=170 HEIGHT=200 SRC="FIGDIR/small/627650v5_ufig1.gif" ALT="Figure 1"> View larger version (65K): org.highwire.dtl.DTLVardef@1873e8org.highwire.dtl.DTLVardef@161a63eorg.highwire.dtl.DTLVardef@1e95b36org.highwire.dtl.DTLVardef@1634ecd_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIA novel murine model in which PKC{varepsilon} is floxed (PKC{varepsilon}fl/fl) on both alleles haas been generated, backcrossed, and deposited into Jackson Laboratories (B6(Cg)-Prkcetm1.1Akv/LennMmjax). Then, PKC{varepsilon}fl/fl LysM Cre (m{varepsilon}KO) mice have been generated. C_LIO_LIDeletion of PKC{varepsilon} has no basal affects on other PKC isoforms, lipid handling markers, or inflammatory markers. C_LIO_LIUpon (in vitro) lipid loading of BMDM or isolating lipid loaded pMACs from hypercholesterolemic mice (in vivo), m{varepsilon}KO BMDMs retain more cholesterol. C_LIO_LILong term (16 wk) hypercholesterolemis generates larger lesions, more necrosis, and thinner plaque caps (i.e., a more vulnerable plaque phenotype) in m{varepsilon}KO mice. C_LIO_LIRNA sequencing suggests PKC{varepsilon} regulates endocytosis mechanisms post initial uptake of LDL and has been confirmed in a model of synchronized endocytosis. C_LIO_LIThese findings identify myeloid PKC{varepsilon} as a novel atheroprotective molecule whose expression impacts multiple metrics of atherosclerosis. This opens new avenues of investigation to uncover the PKC{varepsilon} signaling network responsible for the phenotype. Understanding how myeloid PKC{varepsilon} slows atherosclerosis development will aid in the development of novel preventative and therapeutic treatments. C_LI