Department of Nephrology (T.K., S.M., R.K., H.M., M.C., S.S., T.F., F.A., T.M., K.S., S.I., S.N., E.S., T.R., S.U.), Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Yushima, Bunkyo, Japan.
Department of Human Pathology (K. Yamamoto), Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Yushima, Bunkyo, Japan.
Circ Res. 2023 Feb 17;132(4):415-431. doi: 10.1161/CIRCRESAHA.122.321939. Epub 2023 Jan 26.
BACKGROUND: Chronic kidney disease (CKD) accelerates vascular calcification via phenotypic switching of vascular smooth muscle cells (VSMCs). We investigated the roles of circulating small extracellular vesicles (sEVs) between the kidneys and VSMCs and uncovered relevant sEV-propagated microRNAs (miRNAs) and their biological signaling pathways. METHODS AND RESULTS: We established CKD models in rats and mice by adenine-induced tubulointerstitial fibrosis. Cultures of A10 embryonic rat VSMCs showed increased calcification and transcription of osterix (), osteocalcin (), and osteopontin () when treated with rat CKD serum. sEVs, but not sEV-depleted serum, accelerated calcification in VSMCs. Intraperitoneal administration of a neutral sphingomyelinase and biogenesis/release inhibitor of sEVs, GW4869 (2.5 mg/kg per 2 days), inhibited thoracic aortic calcification in CKD mice under a high-phosphorus diet. GW4869 induced a nearly full recovery of calcification and transcription of osteogenic marker genes. In CKD, the miRNA transcriptome of sEVs revealed a depletion of 4 miRNAs, , cluster-originated /, and . Their expression decreased in sEVs from CKD patients as kidney function deteriorated. Transfection of VSMCs with each miRNA-mimic mitigated calcification. In silico analyses revealed VEGFA (vascular endothelial growth factor A) as a convergent target of these miRNAs. We found a 16-fold increase in transcription in the thoracic aorta of CKD mice under a high-phosphorus diet, which GW4869 reversed. Inhibition of VEGFA-VEGFR2 signaling with sorafenib, fruquintinib, sunitinib, or -targeted siRNA mitigated calcification in VSMCs. Orally administered fruquintinib (2.5 mg/kg per day) for 4 weeks suppressed the transcription of osteogenic marker genes in the mouse aorta. The area under the curve of , , , and for the prediction of abdominal aortic calcification was 0.7630, 0.7704, 0.7407, and 0.7704, respectively. CONCLUSIONS: The miRNA transcriptomic signature of circulating sEVs uncovered their pathologic role, devoid of the calcification-protective miRNAs that target VEGFA signaling in CKD-driven vascular calcification. These sEV-propagated miRNAs are potential biomarkers and therapeutic targets for vascular calcification.
背景：慢性肾脏病（CKD）通过血管平滑肌细胞（VSMC）的表型转换加速血管钙化。我们研究了肾脏和 VSMC 之间循环的小细胞外囊泡（sEVs）的作用，并发现了相关的 sEV 传播 microRNAs（miRNAs）及其生物学信号通路。
方法和结果：我们通过腺嘌呤诱导的肾小管间质纤维化在大鼠和小鼠中建立 CKD 模型。当用大鼠 CKD 血清处理时，A10 胚胎大鼠 VSMC 培养物显示出钙化增加和骨形态发生蛋白 2（osterix，）、骨钙素（osteocalcin，）和骨桥蛋白（osteopontin，）的转录增加。sEVs 而不是不含 sEV 的血清加速了 VSMC 的钙化。腹腔内给予鞘氨醇激酶中和剂和 sEV 生物发生/释放抑制剂 GW4869（每天 2.5mg/kg，每 2 天）可抑制高磷饮食下 CKD 小鼠的胸主动脉钙化。GW4869 诱导了几乎完全恢复的钙化和成骨标记基因的转录。在 CKD 中，sEV 的 miRNA 转录组显示 4 个 miRNA 的耗竭，、簇源性/和。随着肾功能恶化，它们在 CKD 患者的 sEV 中的表达减少。转染 VSMC 中的每个 miRNA 模拟物可减轻钙化。计算机分析显示血管内皮生长因子 A（vascular endothelial growth factor A，VEGFA）是这些 miRNA 的一个收敛靶标。我们发现，在高磷饮食下 CKD 小鼠的胸主动脉中，的转录增加了 16 倍，GW4869 逆转了这一现象。用索拉非尼、呋喹替尼、舒尼替尼或靶向的 siRNA 抑制 VEGFA-VEGFR2 信号可减轻 VSMC 的钙化。连续 4 周口服呋喹替尼（每天 2.5mg/kg）可抑制小鼠主动脉中成骨标记基因的转录。用于预测腹主动脉钙化的、、和的曲线下面积分别为 0.7630、0.7704、0.7407 和 0.7704。
结论：循环 sEV 的 miRNA 转录组特征揭示了它们在 CKD 驱动的血管钙化中的病理作用，缺乏针对 VEGFA 信号的钙化保护 miRNA。这些 sEV 传播的 miRNA 是血管钙化的潜在生物标志物和治疗靶点。
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