Atherosclerotic cardiovascular disease (ASCVD) 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 relevant to ASCVD, and characterize specific monocyte subsets isolated from the circulation of normocholesterolemic controls and hypercholesterolemic patients. We hypothesized that activation of fluid-phase macropinocytosis contributes to foamy monocyte formation in vitro and in hypercholesterolemic mice in vivo. 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. 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. Mechanistic studies identified NADPH oxidase 2 (Nox2)-derived superoxide anion (O2⋅−) as an important downstream signaling molecule stimulating macropinocytosis in monocytes. qRT-PCR identified CD36 as a major scavenger receptor that increases in response to lipid loading in monocytes and deletion of CD36 (Cd36−/−) inhibited foamy monocyte formation in hypercholesterolemic mice. Bulk RNA-sequencing characterized transcriptional differences between non-foamy and foamy monocytes versus macrophages. Finally, 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 for the treatment of atherosclerosis.

中文翻译：

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受体非依赖性液相胞饮作用的激活促进动脉粥样硬化小鼠泡沫状单核细胞的形成


动脉粥样硬化性心血管疾病 （ASCVD） 是全球死亡的主要原因。临床和实验数据表明，循环单核细胞内化血浆脂蛋白，并在高胆固醇血症受试者中成为富含脂质的泡沫细胞。本研究旨在确定负责泡沫状单核细胞形成的内吞机制，对与 ASCVD 相关的泡沫和非泡沫状单核细胞进行功能和转录组学分析，并表征从正常胆固醇血症对照和高胆固醇血症患者的循环中分离的特定单核细胞亚群。我们假设液相巨胞饮作用的激活有助于体外泡沫状单核细胞形成，以及体内高胆固醇血症小鼠的泡沫状单核细胞形成。高分辨率扫描电子显微镜 （SEM） 和 FITC/TRITC-葡聚糖内化的定量表明，人 （THP-1） 和野生型小鼠单核细胞中巨胞饮作用刺激。巨胞饮作用的刺激在体外未修饰的天然 LDL （nLDL） 和氧化 LDL （ox-LDL） 存在下诱导泡沫状单核细胞形成。巨胞饮作用的遗传阻断 （LysMCre+Nhe1f/f） 抑制了高胆固醇血症小鼠体内泡沫状单核细胞的形成，并减弱了单核细胞对体外动脉粥样硬化主动脉的粘附。机制研究确定 NADPH 氧化酶 2 （Nox2） 衍生的超氧阴离子 （O2⋅−） 是刺激单核细胞巨胞饮作用的重要下游信号分子。qRT-PCR 发现 CD36 是一种主要的清道夫受体，它响应单核细胞中的脂质负荷而增加，CD36 （Cd36−/−） 的缺失抑制了高胆固醇血症小鼠的泡沫状单核细胞形成。 大量 RNA 测序表征了非泡沫和泡沫单核细胞与巨噬细胞之间的转录差异。最后，CD14 和 CD16 表达的流式细胞术分析表明，与正常胆固醇血症对照相比，高胆固醇血症患者的中间单核细胞显著增加。这些结果为泡沫状单核细胞形成的机制提供了新的见解，并可能确定治疗动脉粥样硬化的新治疗靶点。