Bobek Jordan M, Stuttgen Gage M, Sahoo Daisy
Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI, United States.
Cardiovascular Center, Medical College of Wisconsin, Milwaukee, WI, United States.
Front Cardiovasc Med. 2024 Sep 12;11:1448607. doi: 10.3389/fcvm.2024.1448607. eCollection 2024.
Recent findings demonstrate that high density lipoprotein (HDL) function rather than HDL-cholesterol levels themselves may be a better indicator of cardiovascular disease risk. One mechanism by which HDL can become dysfunctional is through oxidative modification by reactive aldehydes. Previous studies from our group demonstrated that HDL modified by reactive aldehydes alters select cardioprotective functions of HDL in macrophages. To identify mechanisms by which dysfunctional HDL contributes to atherosclerosis progression, we designed experiments to test the hypothesis that HDL modified by reactive aldehydes triggers endoplasmic reticulum (ER) stress in primary murine macrophages.
Peritoneal macrophages were harvested from wild-type C57BL/6J mice and treated with thapsigargin, oxLDL, and/or HDL for up to 48 hours. Immunoblot analysis and semi-quantitative PCR were used to measure expression of BiP, p-eIF2α, ATF6, and XBP1 to assess activation of the unfolded protein response (UPR). Through an extensive set of comprehensive experiments, and contrary to some published studies, our findings led us to three novel discoveries in primary murine macrophages: (i) oxLDL alone was unable to induce ER stress; (ii) co-incubation with oxLDL or HDL in the presence of thapsigargin had an additive effect in which expression of ER stress markers were significantly increased and prolonged as compared to cells treated with thapsigargin alone; and (iii) HDL, in the presence or absence of reactive aldehydes, was unable blunt the ER stress induced by thapsigargin in the presence or absence of oxLDL.
Our systematic approach to assess the role of native and modified HDL in mediating primary macrophage ER stress led to the discovery that lipoproteins on their own require the presence of thapsigargin to synergistically increase expression of ER stress markers. We further demonstrated that HDL, in the presence or absence of reactive aldehydes, was unable to blunt the ER stress induced by thapsigargin in the presence or absence of oxLDL. Together, our findings suggest the need for more detailed investigations to better understand the role of native and modified lipoproteins in mediating ER stress pathways.
最近的研究结果表明，高密度脂蛋白（HDL）的功能而非HDL胆固醇水平本身可能是心血管疾病风险的更好指标。HDL功能失调的一种机制是通过活性醛的氧化修饰。我们团队之前的研究表明，经活性醛修饰的HDL会改变巨噬细胞中HDL的某些心脏保护功能。为了确定功能失调的HDL促进动脉粥样硬化进展的机制，我们设计了实验来检验以下假设：经活性醛修饰的HDL会在原代小鼠巨噬细胞中引发内质网（ER）应激。
从野生型C57BL/6J小鼠中收集腹腔巨噬细胞，并用毒胡萝卜素、氧化型低密度脂蛋白（oxLDL）和/或HDL处理长达48小时。采用免疫印迹分析和半定量PCR来测量结合免疫球蛋白重链结合蛋白（BiP）、磷酸化真核翻译起始因子2α（p-eIF2α）、活化转录因子6（ATF6）和X盒结合蛋白1（XBP1）的表达，以评估未折叠蛋白反应（UPR）的激活情况。通过一系列广泛的综合实验，与一些已发表的研究结果相反，我们的研究结果在原代小鼠巨噬细胞中带来了三个新发现：（i）单独的oxLDL无法诱导ER应激；（ii）在毒胡萝卜素存在的情况下，与oxLDL或HDL共同孵育具有累加效应，与单独用毒胡萝卜素处理的细胞相比，ER应激标志物的表达显著增加且持续时间延长；（iii）无论是否存在活性醛，HDL都无法在有或没有oxLDL的情况下减弱毒胡萝卜素诱导的ER应激。
我们评估天然和修饰HDL在介导原代巨噬细胞ER应激中作用的系统方法导致发现，脂蛋白本身需要毒胡萝卜素的存在才能协同增加ER应激标志物的表达。我们进一步证明，无论是否存在活性醛，HDL都无法在有或没有oxLDL的情况下减弱毒胡萝卜素诱导的ER应激。总之，我们的研究结果表明需要进行更详细的研究，以更好地了解天然和修饰脂蛋白在介导ER应激途径中的作用。
