Plaque-targeted, proteolysis-resistant, activatable and MRI-visible nano-GLP-1 receptor agonist targets smooth muscle cell differentiation in atherosclerosis.
Plaque-targeted, proteolysis-resistant, activatable and MRI-visible nano-GLP-1 receptor agonist targets smooth muscle cell differentiation in atherosclerosis.
复制标题
DOI：
10.7150/thno.66456
复制
发表时间：
2022
期刊：
影响因子：
12.4
通讯作者：
Rajagopalan S
中科院分区：
文献类型：
--
作者：
Maiseyeu A;Di L;Ravodina A;Barajas-Espinosa A;Sakamoto A;Chaplin A;Zhong J;Gao H;Mignery M;Narula N;Finn AV;Rajagopalan S
背景：胰高血糖素样多肽-1受体(GLP-1R)激动剂是一种有效的降血糖药物，已被系统地证明可降低心血管事件和死亡率。由于这些激动剂对血管细胞缺乏特殊的特异性，而且对GLP-1R在动脉粥样硬化中的表达缺乏充分的了解，这些有益的影响很难在动脉粥样硬化斑块中准确地确定。在这里，我们假设GLP-1R通过靶向激动剂直接参与动脉粥样硬化将减轻血管炎症和斑块负担，即使在非常低的剂量下也是如此。方法：采用基因谱系追踪、Southern杂交和抗GLP-1R抗血清检测GLP-1受体(GLP-1R、GLP1R)在人病理性内膜增厚、APOE-/-小鼠动脉粥样硬化和培养的免疫/非免疫细胞中的表达。设计并合成了携带GLP-1R激动剂利拉鲁肽(GlpNP)的抗酶活性纳米颗粒(NPs)。通过核磁共振成像，在GlpNP中包合了Gd螯合物。给动脉粥样硬化APOE-/-小鼠单次静脉注射利拉鲁肽(30微克/公斤)或长期给予GlpNP、利拉鲁肽或对照纳米粒(1微克/公斤、6周、2倍/周)，然后评估代谢参数、动脉粥样硬化负荷、炎症反应和血管功能。结果：人类斑块标本在去分化的平滑肌细胞中高水平表达GLP-1R，该细胞也表达髓系标志CD68。然而，先天免疫细胞在各种条件下表达非常低水平的GLP1R，正如谱系追踪和Southern blotting实验检测全长开放阅读框架mRNA转录本所看到的那样。重要的是，去分化的血管平滑肌细胞表现出显著的GLP1R表达水平，这表明这些细胞可能代表动脉粥样硬化中以GLP1R阳性为主的细胞。GlpNP具有抗蛋白质降解和生物活性，包括体内降血糖30微克/公斤和体外胆固醇流出。GlpNP的激活特性在体外通过成像细胞术得到证实，在体内通过整体器官成像得到证实。通过MRI和荧光成像评估GlpNP靶向APOE-/-小鼠循环中的CD11b+/CD11c+细胞和主动脉斑块中的平滑肌细胞。GlpNP静脉注射的剂量非常低，为1微克/公斤，以前已知对血糖和体重减轻几乎没有影响。在六周内降低血浆中富含甘油三酯的脂蛋白、斑块负担和斑块胆固醇，而对体重、血糖和血浆胆固醇水平没有显著影响。结论：GlpNP在体重中性剂量低至1微克/公斤时可改善动脉粥样硬化，其作用不依赖于胰腺或中枢神经系统。我们的研究强调了GLP-1类似物在动脉粥样硬化中直接作用的重要性，包括胆固醇外流和炎症。我们的发现首次表明GlpNP对血管靶点的治疗调节，特别是在平滑肌细胞炎症的背景下。
Background: Glucagon-like peptide-1 receptor (GLP-1R) agonists are powerful glycemia-lowering agents, which have systematically been shown to lower cardiovascular events and mortality. These beneficial effects were difficult to pinpoint within atherosclerotic plaque due to lack of particular specificity of such agonists to the vascular cells and an inadequate understanding of the GLP-1R expression in atherosclerosis. Here, we hypothesized that the direct engagement of the GLP-1R in atherosclerosis by targeted agonists will alleviate vascular inflammation and plaque burden, even at a very low dose. Methods: The expression of GLP-1 receptor (GLP-1R, Glp1r mRNA) in human lesions with pathologic intimal thickening, Apoe-/- mouse atheroma and cultured immune/non-immune cells was investigated using genetic lineage tracing, Southern blotting and validated antisera against human GLP-1R. Protease-resistant and “activatable” nanoparticles (NPs) carrying GLP-1R agonist liraglutide (GlpNP) were engineered and synthesized. Inclusion of gadolinium chelates into GlpNP allowed for imaging by MRI. Atherosclerotic Apoe-/- mice were treated intravenously with a single dose (30 µg/kg of liraglutide) or chronically (1 µg/kg, 6 weeks, 2x/week) with GlpNP, liraglutide or control NPs, followed by assessment of metabolic parameters, atheroma burden, inflammation and vascular function. Results: Humal plaque specimens expressed high levels of GLP-1R within the locus of de-differentiated smooth muscle cells that also expressed myeloid marker CD68. However, innate immune cells under a variety of conditions expressed very low levels of Glp1r, as seen in lineage tracing and Southern blotting experiments examining full-length open reading frame mRNA transcripts. Importantly, de-differentiated vascular smooth muscle cells demonstrated significant Glp1r expression levels, suggesting that these could represent the cells with predominant Glp1r-positivity in atherosclerosis. GlpNP resisted proteolysis and demonstrated biological activity including in vivo glycemia lowering at 30 µg/kg and in vitro cholesterol efflux. Activatable properties of GlpNP were confirmed in vitro by imaging cytometry and in vivo using whole organ imaging. GlpNP targeted CD11b+/CD11c+ cells in circulation and smooth muscle cells in aortic plaque in Apoe-/- mice when assessed by MRI and fluorescence imaging. At a very low dose of 1 µg/kg, previously known to have little effect on glycemia and weight loss, GlpNP delivered i.v. for six weeks reduced triglyceride-rich lipoproteins in plasma, plaque burden and plaque cholesterol without significant effects on weight, glycemia and plasma cholesterol levels. Conclusions: GlpNP improves atherosclerosis at weight-neutral doses as low as 1 µg/kg with the effects independent from the pancreas or the central nervous system. Our study underlines the importance of direct actions of GLP-1 analogs on atherosclerosis, involving cholesterol efflux and inflammation. Our findings are the first to suggest the therapeutic modulation of vascular targets by GlpNP, especially in the context of smooth muscle cell inflammation.