{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,1,15]],"date-time":"2026-01-15T22:29:36Z","timestamp":1768516176056,"version":"3.49.0"},"posted":{"date-parts":[[2024,12,14]]},"group-title":"Immunology","reference-count":68,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2024,12,14]],"date-time":"2024-12-14T00:00:00Z","timestamp":1734134400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/www.biorxiv.org\/about\/FAQ#license"}],"funder":[{"name":"NIH","award":["1R21AI185827"],"award-info":[{"award-number":["1R21AI185827"]}]},{"name":"NIH","award":["R03 AI144841"],"award-info":[{"award-number":["R03 AI144841"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"abstract":"<jats:sec>\n                  <jats:title>BACKGROUND<\/jats:title>\n                  <jats:p>Atherosclerosis is a chronic inflammatory disease driven by macrophages (M\u00d8). Protein Kinase C \u2013 epsilon (PKC\u025b) is a serine\/threonine kinase involved in diverse cellular processes including migration, growth, differentiation, and survival. PKC\u025b acts in a context dependent manner within the heart, however, its role in atherosclerosis is unknown.<\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>METHODS<\/jats:title>\n                  <jats:p>\n                    Bone marrow derived M\u00d8 from global PKC\u025b 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\u025b\n                    <jats:sup>fl\/fl<\/jats:sup>\n                    (m\u025bKO) mouse was developed to study the impact of myeloid PKC\u025b on atherosclerosis. After confirming myeloid selective PKC\u025b deletion, human-like hypercholesterolemia was induced and multiple metrics of atherosclerosis were compared in WT and m\u025bKO plaques. RNA sequencing was used to provide unbiased insight into possible mechanisms by which PKC\u025b regulates atherosclerosis.\n                  <\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>RESULTS<\/jats:title>\n                  <jats:p>Public geneset analysis of human atherosclerotic plaque tissue revealed that PKC\u025b expression is inversely correlated with plaque vulnerability. Similarly, peritoneal M\u00d8 from WT hypercholesterolemic mice have significantly lower PKC\u025b expression, providing a translational rational for generation of the m\u025bKO 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\u025b 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\u025bKO hypercholesterolemic mice revealed that m\u025bKO plaques are larger, have larger foam cells and regions of necrosis, and thinner collagen caps. Upon lipid loading in vitro and in vivo, m\u025bKO M\u00d8 retained significantly more cholesterol and lipid droplets than WT; Gene Ontology suggests higher expression of genes related to endocytosis in m\u025bKO M\u00d8 compared to WT.<\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>CONCLUSIONS<\/jats:title>\n                  <jats:p>PKC\u025b expression is decreased in vulnerable human plaques and decreases in mouse M\u00d8 upon lipid loading. m\u025bKO plaques are larger and exhibit markers of vulnerability. With no differences in scavenger receptor (SR) expression, the impact of PKC\u025b deletion is more subtle than simple SR dysregulation. RNA sequencing implicates higher expression of genes involved in endocytosis and m\u025bKO M\u00d8 have significantly more lipid-containing endosomes. The data define the atherophenotype of m\u025bKO mice and demonstrate that PKC\u025b restricts lipid uptake into M\u00d8 by a mechanism independent of SR expression. Taken together, these studies identify PKC\u025b as a novel atheroprotective gene, laying the foundation for mechanistic studies on the endocytic signaling networks responsible for the phenotype.<\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>GRAPHIC ABSTRACT<\/jats:title>\n                  <jats:fig id=\"ufig1\" position=\"float\" orientation=\"portrait\" fig-type=\"figure\">\n                    <jats:graphic xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" xlink:href=\"627650v5_ufig1\" position=\"float\" orientation=\"portrait\"\/>\n                  <\/jats:fig>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Highlights<\/jats:title>\n                  <jats:list list-type=\"bullet\">\n                    <jats:list-item>\n                      <jats:p>\n                        A novel murine model in which PKC\u03b5 is floxed (PKC\u03b5\n                        <jats:sup>fl\/fl<\/jats:sup>\n                        ) on both alleles haas been generated, backcrossed, and deposited into Jackson Laboratories (B6(Cg)-\n                        <jats:italic>\n                          Prkce\n                          <jats:sup>tm1.1Akv<\/jats:sup>\n                        <\/jats:italic>\n                        \/LennMmjax). Then, PKC\u03b5\n                        <jats:sup>fl\/fl<\/jats:sup>\n                        LysM Cre (m\u03b5KO) mice have been generated.\n                      <\/jats:p>\n                    <\/jats:list-item>\n                    <jats:list-item>\n                      <jats:p>Deletion of PKC\u03b5 has no basal affects on other PKC isoforms, lipid handling markers, or inflammatory markers.<\/jats:p>\n                    <\/jats:list-item>\n                    <jats:list-item>\n                      <jats:p>Upon (in vitro) lipid loading of BMDM or isolating lipid loaded pMACs from hypercholesterolemic mice (in vivo), m\u03b5KO BMDMs retain more cholesterol.<\/jats:p>\n                    <\/jats:list-item>\n                    <jats:list-item>\n                      <jats:p>Long term (16 wk) hypercholesterolemis generates larger lesions, more necrosis, and thinner plaque caps (i.e., a more vulnerable plaque phenotype) in m\u03b5KO mice.<\/jats:p>\n                    <\/jats:list-item>\n                    <jats:list-item>\n                      <jats:p>RNA sequencing suggests PKC\u03b5 regulates endocytosis mechanisms post initial uptake of LDL and has been confirmed in a model of synchronized endocytosis.<\/jats:p>\n                    <\/jats:list-item>\n                    <jats:list-item>\n                      <jats:p>These findings identify myeloid PKC\u03b5 as a novel atheroprotective molecule whose expression impacts multiple metrics of atherosclerosis. This opens new avenues of investigation to uncover the PKC\u03b5 signaling network responsible for the phenotype. Understanding how myeloid PKC\u03b5 slows atherosclerosis development will aid in the development of novel preventative and therapeutic treatments.<\/jats:p>\n                    <\/jats:list-item>\n                  <\/jats:list>\n                <\/jats:sec>","DOI":"10.1101\/2024.12.09.627650","type":"posted-content","created":{"date-parts":[[2024,12,14]],"date-time":"2024-12-14T10:25:18Z","timestamp":1734171918000},"source":"Crossref","is-referenced-by-count":0,"title":["Identification of Myeloid Protein Kinase C \u2013 Epsilon as a Novel Atheroprotective Gene"],"prefix":"10.64898","author":[{"given":"Alexis T.","family":"Wells","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8536-2606","authenticated-orcid":false,"given":"Ramon Bossardi","family":"Ramos","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Michelle M.","family":"Shen","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Redwan H.","family":"Binrouf","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Anna E.","family":"D\u2019Amico","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2519-8805","authenticated-orcid":false,"given":"Michelle R.","family":"Lennartz","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"54368","reference":[{"key":"2025050504000481000_2024.12.09.627650v5.1","unstructured":"What is Cardiovascular Disease. https:\/\/www.heart.org\/en\/health-topics\/consumer-healthcare\/what-is-cardiovascular-disease."},{"key":"2025050504000481000_2024.12.09.627650v5.2","doi-asserted-by":"publisher","DOI":"10.5334\/gh.1288"},{"key":"2025050504000481000_2024.12.09.627650v5.3","unstructured":"Heart Disease Facts. 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