Macropinocytosis promotes foamy monocyte formation in hypercholesterolemic mice

Atherosclerosis-associated vascular disease is the leading cause of death worldwide. Clinical and experimental data demonstrated that circulating monocytes internalize plasma lipoproteins and become lipid-laden foamy cells in hypercholesterolemic subjects. This study was designed to identify the endocytic mechanisms responsible for foamy monocyte formation, perform functional and transcriptomic analysis of foamy and non-foamy monocytes, and characterize specific monocyte subsets in the circulation from normocholesterolemic controls and hypercholesterolemic patients. The presence of foamy monocytes was confirmed in hypercholesterolemic mice and humans via flow cytometry analysis. High resolution scanning electron microscopy (SEM) and quantification of FITC/TRITC-dextran internalization demonstrated macropinocytosis stimulation in human (THP-1) and wild type murine monocytes in vitro. Stimulation of macropinocytosis induced foamy monocyte formation in the presence of unmodified, native LDL (nLDL) and oxidized LDL (ox-LDL) in vitro. Genetic blockade of macropinocytosis (LysMCre+ Nhe1f/f) inhibited foamy monocyte formation in hypercholesterolemic mice in vivo and attenuated monocyte adhesion to atherosclerotic aortas ex vivo. qRT-PCR quantified mRNA levels of major scavenger receptors (SR) in foamy and non-foamy monocytes and identified CD36 as a major SR increasing in response to lipid loading. Deletion of CD36 (Cd36-/-) inhibited foamy monocyte formation in hypercholesterolemic mice. Mechanistic studies identified NADPH oxidase 2 (Nox2)-derived superoxide (O2·-) as an important downstream signaling molecule stimulating macropinocytosis in monocytes. Bulk RNA-sequencing characterized transcriptional differences between non-foamy and foamy monocytes and macrophages. Flow cytometry analysis of CD14 and CD16 expression demonstrated a significant increase in intermediate monocytes in hypercholesterolemic patients compared to normocholesterolemic controls. These results provide novel insights into the mechanisms of foamy monocyte formation and potentially identify new therapeutic targets in the treatment of atherosclerosis. Overall design: To investigate differential gene expression in foamy and non-foamy monocytes and macrophages, primary splenic monocytes were isolated from wild type C57BL/6 mice and differentiated into macrophages. Cells were treated with vehicle or ox-LDL. Bulk RNA sequencing was performed to assess transcriptional changes.

动脉粥样硬化相关血管疾病是全球首要致死病因。临床与实验研究证实，高胆固醇血症患者体内的循环单核细胞可内吞血浆脂蛋白，进而转变为脂质过载的泡沫细胞。本研究旨在明确泡沫单核细胞形成的内吞机制，对泡沫与非泡沫单核细胞开展功能及转录组分析，并对正常胆固醇血症对照个体与高胆固醇血症患者循环中的特定单核细胞亚群进行表征。研究通过流式细胞术证实，高胆固醇血症小鼠及人体内均存在泡沫单核细胞。高分辨率扫描电子显微镜（SEM）检测及异硫氰酸荧光素/四甲基罗丹明标记葡聚糖（FITC/TRITC-dextran）内吞定量实验证实，体外培养的人源THP-1细胞与野生型小鼠单核细胞中巨胞饮作用被激活。在天然未修饰低密度脂蛋白（nLDL）与氧化低密度脂蛋白（ox-LDL）存在的条件下，巨胞饮作用的激活可在体外诱导泡沫单核细胞形成。通过基因手段阻断巨胞饮作用（LysMCre+ Nhe1f/f），可在体内抑制高胆固醇血症小鼠的泡沫单核细胞形成，并在离体实验中减弱单核细胞与动脉粥样硬化主动脉的黏附能力。实时定量逆转录聚合酶链反应（qRT-PCR）对泡沫与非泡沫单核细胞中主要清道夫受体（SR）的mRNA水平进行定量分析，发现CD36是响应脂质负载而上调的核心清道夫受体。敲除CD36基因（Cd36-/-）可抑制高胆固醇血症小鼠体内的泡沫单核细胞形成。机制研究证实，烟酰胺腺嘌呤二核苷酸磷酸氧化酶2（Nox2）介导产生的超氧阴离子（O2·-）是激活单核细胞巨胞饮作用的关键下游信号分子。批量RNA测序对非泡沫与泡沫单核细胞及巨噬细胞之间的转录组差异进行了系统表征。对CD14与CD16表达的流式细胞术分析显示，与正常胆固醇血症对照个体相比，高胆固醇血症患者体内的中间型单核细胞占比显著升高。上述研究结果为泡沫单核细胞的形成机制提供了全新视角，并有望为动脉粥样硬化的治疗发掘新的潜在治疗靶点。整体实验设计：为探究泡沫与非泡沫单核细胞及巨噬细胞的差异基因表达情况，研究人员从野生型C57BL/6小鼠体内分离原代脾脏单核细胞并诱导分化为巨噬细胞。将细胞以溶剂对照或ox-LDL处理后，开展批量RNA测序以评估转录组变化。