Department of Biomedical Engineering, School of Medicine and School of Engineering, University of Alabama at Birmingham, Birmingham, Alabama, USA.
Department of Medicine, Division of Cardiovascular Disease, School of Medicine, University of Alabama at Birmingham, Birmingham, Alabama, USA.
J Extracell Vesicles. 2022 Oct;11(10):e12246. doi: 10.1002/jev2.12246.
Toxoplasma gondii uracil phosphoribosyltransferase (UPRT) converts 4-thiouracil (4TUc) into 4-thiouridine (4TUd), which is incorporated into nascent RNAs and can be biotinylated, then labelled with streptavidin conjugates or isolated via streptavidin-affinity methods. Here, we generated mice that expressed T. gondii UPRT only in cardiomyocytes ( UPRT mice) and tested our hypothesis that CM-derived miRNAs ( miRs) are transferred into remote organs after myocardial infarction (MI) by small extracellular vesicles (sEV) that are released from the heart into the peripheral blood ( sEV). We found that 4TUd was incorporated with high specificity and sensitivity into RNAs isolated from the hearts and sEV of UPRT mice 6 h after 4TUc injection. In sEV, 4TUd was incorporated into CM-specific/enriched miRs including miR-208a, but not into miRs with other organ or tissue-type specificities. 4TUd-labelled miR208a was also present in lung tissues, especially lung endothelial cells (ECs), and CM-derived miR-208a ( miR-208a) levels peaked 12 h after experimentally induced MI in sEV and 24 h after MI in the lung. Notably, miR-208a is expressed from intron 29 of α myosin heavy chain (αMHC), but αMHC transcripts were nearly undetectable in the lung. When sEV from mice that underwent MI (MI- sEV) or sham surgery (Sham- sEV) were injected into intact mice, the expression of Tmbim6 and NLK, which are suppressed by miR-208a and cooperatively regulate inflammation via the NF-κB pathway, was lower in the lungs of MI- sEV-treated animals than the lungs of animals treated with Sham- sEV or saline. In MI mice, Tmbim6 and NLK were downregulated, whereas endothelial adhesion molecules and pro-inflammatory cells were upregulated in the lung; these changes were significantly attenuated when the mice were treated with miR-208a antagomirs prior to MI surgery. Thus, UPRT mice enables us to track sEV-mediated transport of miRs and identify an miR-208a-mediated mechanism by which myocardial injury alters the expression of genes and inflammatory response in the lung.
刚地弓形虫尿嘧啶磷酸核糖基转移酶（UPRT）将 4-硫代尿嘧啶（4TUc）转化为 4-硫代尿苷（4TUd），4TUd 掺入新生 RNA 中，并可被生物素化，然后用链霉亲和素缀合物标记或通过链霉亲和素亲和方法分离。在这里，我们生成了仅在心肌细胞中表达弓形虫 UPRT 的小鼠（UPRT 小鼠），并测试了我们的假设，即心肌梗死后（MI），来自心脏的小细胞外囊泡（sEV）将心脏来源的 microRNA（miRs）转移到远处器官。我们发现，4TUd 特异性和敏感性地掺入了 UPRT 小鼠注射 4TUc 6 小时后心脏和 sEV 中分离的 RNA。在 sEV 中，4TUd 掺入到包括 miR-208a 在内的 CM 特异性/富集的 miRs 中，但不掺入具有其他器官或组织特异性的 miRs 中。4TUd 标记的 miR208a 也存在于肺组织中，特别是肺内皮细胞（ECs）中，并且在实验诱导的 MI 后 12 小时 sEV 和 MI 后 24 小时肺中 CM 来源的 miR-208a（miR-208a）水平达到峰值。值得注意的是，miR-208a 由肌球蛋白重链 α 基因（αMHC）的内含子 29 表达，但在肺中几乎检测不到 αMHC 转录本。当注射心肌梗死后（MI-sEV）或假手术（Sham-sEV）小鼠的 sEV 时，接受 MI-sEV 治疗的动物的肺中 Tmbim6 和 NLK 的表达低于接受 Sham-sEV 或生理盐水治疗的动物的肺，Tmbim6 和 NLK 是受 miR-208a 抑制并通过 NF-κB 途径协同调节炎症的基因。在 MI 小鼠中，Tmbim6 和 NLK 下调，而内皮黏附分子和促炎细胞在肺中上调；在 MI 手术前用 miR-208a 反义寡核苷酸治疗时，这些变化明显减轻。因此，UPRT 小鼠使我们能够追踪 sEV 介导的 miR 转移，并确定心肌损伤改变肺中基因表达和炎症反应的 miR-208a 介导机制。
