Circulating Extracellular Vesicle-Propagated microRNA Signature as a Vascular Calcification Factor in Chronic Kidney Disease

Abstract

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 ( Sp7 ), osteocalcin ( Bglap ), and osteopontin ( Spp1 ) 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, miR-16-5p , miR-17~92 cluster-originated miR-17-5p / miR-20a-5p , and miR-106b-5p . 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 VEGFA 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 VEGFR2 -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 miR-16-5p , miR-17-5p , 20a-5p , and miR-106b-5p 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.

Blockade of exosome release alleviates the hypersensitive reaction by influencing the T helper cell population in cow's milk allergic mice

“…[24][25][26][27][28] It was reported that blocking the release of exosomes with GW4869 suppressed several processes of diseases, including atrial fibrillation, kidney disease, chronic airway disease, ulcerative colitis, and ischemia-reperfusion injury. 26,[29][30][31] In our study, we first constructed a BLG-induced CMA mouse model and set the addition of an exosome release inhibitor (GW4869) group. Female mice were used in our study because they exhibited more active Th2 cell activity than male mice.…”

Exosomes from senescent epithelial cells activate pulmonary fibroblasts via the miR-217-5p/Sirt1 axis in paraquat-induced pulmonary fibrosis

“…For the induction of pulmonary fibrosis [ 19 ], mice were administered intraperitoneal injections of either 20 mg/kg PQ in 100 μl saline (n = 6) or an equivalent volume of saline as a vehicle control (n = 6). For the purpose of treating PQ-induced pulmonary fibrosis, mice were administered GW4869 (2.5 mg/kg) via intraperitoneal injection twice a week [ 20 ], or received intratracheal injection of miR-217-5p antagomir on day 3 following PQ treatment. The lung tissues were collected on day 21 after PQ treatment for subsequent investigation.…”

Vascular calcification: from the perspective of crosstalk

“…GW4869 is a non-competitive neutral sphingomyelinase inhibitor that prevents ILVs formation capable of blocking exosome production [ 63 – 66 ]. Several studies have shown that calcification can be significantly ameliorated after the use of GW4869 at the cellular level or in mice models [ 67 – 69 ], or that GW4869 partially reverses substances that act as inhibitors of calcification [ 70 , 71 ]. These two reviews provide a detailed assessment of the major drugs currently interfering with the biosynthesis or release of EVs [ 65 , 72 ].…”

“…Guava FACS analysis of EV from dialysis patients at T0 showed that only endothelium-derived EVs were up-regulated among most circulating EVs derived from monocytes/macrophages, platelets and endothelial cells in CKD patients compared with healthy recipients, indicating that endothelial cells are the main participants in circulating EVs in CKD patients [ 259 ]. A recent study suggests that by transcriptomic analysis of circulating sEVs from CKD mice models as well as from CKD patients, possibly from endothelial cells, the calcification-protecting miRNAs that target VEGFA signaling in CKD-driven vascular calcification are lacking: miR-16-5p, miR-17-5p, miR-20a-5p, and miR-106b-5p [ 67 ]. And the sEVs biogenesis system inhibitor GW4869 [ 63 – 65 ], was used to ameliorate aortic VC in CKD model mice [ 67 ].…”