Wells Alexis T, Shen Michelle M, Binrouf Redwan H, D'Amico Anna E, Bossardi Ramos Ramon, Lennartz Michelle R
bioRxiv. 2024 Dec 18:2024.12.09.627650. doi: 10.1101/2024.12.09.627650.
Atherosclerosis is a lipid mediated chronic inflammatory disease driven my macrophages (MØ). Protein Kinase C - epsilon (PKCɛ) is is a serine/threonine kinase involved in diverse cellular processes such as migration, growth, differentiation, and survival. PKCɛ is known to act in a context dependent manner within heart, however, its role in atherosclerosis is unknown.
Bone marrow derived MØ from global PKCɛ KO mice were examined for impact of lipid metabolism and inflammatory factor secretion. Public geneset analysis assessed raw counts of PKCɛ to determine translational relevance. To determine the function myeloid PKCɛ on atherosclerosis a novel murine model was generated using LysM Cre technology. After its characterization, human-like hypercholesterolemia was induced to assess plaque morphology in WT mice or mice lacking myeloid PKCɛ.
Public geneset analysis of human atherosclerotic plaque tissue revealed that PKCɛ expression is inversely correlated with plaque size and vulnerability. Similarly, peritoneal MØ from hypercholesterolemic mice have significantly lower PKCɛ expression. As MØ play a major role in atherogenesis, we generated a mouse strain with PKCɛ selectively deleted in the myeloid lineage (mɛKO). qPCR revealed no basal differences between genotypes in the expression of lipid uptake receptors, efflux transporters, or inflammatory markers. However, upon lipid loading, mɛKO MØs retained significantly more cholesterol than WT. Human-like hypercholesterolemia was induced in WT and mɛKO mice and assessed for lesion area and plaque morphology in aortic arches and aortic roots. We found that, compared to WT, the lesion area in mɛKO mice was significantly larger, more necrotic, had larger foam cells, and thinner collagen caps.
Loss of myeloid PKCɛ promotes atherosclerosis as determined by larger lesions, more necrosis, thinner plaque caps). Together, these data identify myeloid PKCɛ as a novel atheroprotective gene, laying the foundation for mechanistic studies on the signaling networks responsible for the phenotype.
A novel murine model in which PKCɛ is floxed (PKCɛ ) on both alleles haas been generated, backcrossed, and deposited into Jackson Laboratories. PKCε mice have been crossed with those on the LysM Cre background thereby deleting PKCε from myeloid cells (mεKO). Deletion of PKCε has no basal affects on other PKC isoforms, lipid handling markers, or inflammatory markers.Upon stimulation with lopid loading in vitro or hypercholesterolemia in vivo, mεKO BMDMs retain more cholesterol and mεKO mice develop a more vulnerable plaque phenotype (i.e. larger lesions, more necrosis, thimmer plaque caps).These findings provide a rationale for the need to identify mediators in the PKCε signaling pathway responsible for protection against vulnerable plaques in atherosclerosis; potentially aiding in the development of preventative and therapeutic treatments.
动脉粥样硬化是一种由巨噬细胞（MØ）驱动的脂质介导的慢性炎症性疾病。蛋白激酶C-ε（PKCɛ）是一种丝氨酸/苏氨酸激酶，参与多种细胞过程，如迁移、生长、分化和存活。已知PKCɛ在心脏中以依赖于上下文的方式发挥作用，然而，其在动脉粥样硬化中的作用尚不清楚。
检测来自全球PKCɛ基因敲除小鼠的骨髓源性MØ对脂质代谢和炎症因子分泌的影响。公共基因集分析评估PKCɛ的原始计数以确定翻译相关性。为了确定髓系PKCɛ在动脉粥样硬化中的功能，使用LysM Cre技术建立了一种新型小鼠模型。在对其进行表征后，诱导产生类似人类的高胆固醇血症，以评估野生型小鼠或缺乏髓系PKCɛ的小鼠的斑块形态。
对人类动脉粥样硬化斑块组织的公共基因集分析表明，PKCɛ表达与斑块大小和易损性呈负相关。同样，高胆固醇血症小鼠的腹腔MØ中PKCɛ表达明显较低。由于MØ在动脉粥样硬化形成中起主要作用，我们构建了一种在髓系谱系中选择性缺失PKCɛ的小鼠品系（mɛKO）。qPCR显示，在脂质摄取受体、流出转运蛋白或炎症标志物的表达方面，各基因型之间没有基础差异。然而，在脂质加载后，mɛKO MØ比野生型保留了更多的胆固醇。在野生型和mɛKO小鼠中诱导产生类似人类的高胆固醇血症，并评估主动脉弓和主动脉根部的病变面积和斑块形态。我们发现，与野生型相比，mɛKO小鼠的病变面积明显更大，坏死更多，泡沫细胞更大，胶原帽更薄。
髓系PKCɛ的缺失促进了动脉粥样硬化，表现为病变更大、坏死更多、斑块帽更薄。总之，这些数据确定髓系PKCɛ是一种新型的抗动脉粥样硬化基因，为负责该表型的信号网络的机制研究奠定了基础。
已经构建了一种新型小鼠模型，其中两个等位基因上的PKCɛ都被敲除（PKCɛ ），回交并保存在杰克逊实验室。PKCε 小鼠已与LysM Cre背景的小鼠杂交，从而从髓系细胞中删除PKCε（mεKO）。PKCε的缺失对其他PKC同工型、脂质处理标志物或炎症标志物没有基础影响。在体外脂质加载或体内高胆固醇血症刺激后，mεKO骨髓来源的巨噬细胞（BMDM）保留更多胆固醇，mεKO小鼠形成更易损的斑块表型（即病变更大、坏死更多、斑块帽更薄）。这些发现为确定PKCε信号通路中负责预防动脉粥样硬化中易损斑块的介质提供了理论依据；可能有助于预防和治疗方法的开发。
