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. Author manuscript; available in PMC: 2021 Aug 14.
Published in final edited form as: Circ Res. 2020 Jun 4;127(5):677–692. doi: 10.1161/CIRCRESAHA.119.316398

Figure 2. Chronic conjoining of the circulatory systems in mice via parabiosis leads to delivery of blood-borne miR-210 to pulmonary arterial endothelial cells in vivo.

Figure 2.

(A) An in vivo parabiosis platform allowed for the conjoining of the circulatory systems of two mice in the context of miR-210 +/+ (WT) and miR-210 −/− (KO) partners. One-week post-surgery, mouse pairs were exposed to chronic hypoxia (10% O2) for 3 weeks to induce endogenous miR-210 up-regulation. (Red arrows denote comparison groups.) (B-D) As assessed by RT-qPCR in plasma (B, N=8,7,9,8 mice) and in CD31+/CD45− lung endothelial cells (D, N=12,11,10,12 mice), while miR-210 expression was negligible in KO-KO pairs, extracellular miR-210 level in KO partners of KO-WT pairs was increased to comparable level as WT-WT pairs whereas intracellular miR-210 in those mice was readily detected but still less than that of WT partners and WT-WT pairs. Extracellular miR-210 in plasma was found to be co-immunoprecipitated specifically in the presence of α-Argonaute 2 (AGO2) as compared with control IgG (C, N=4 mice/group). (E) By in situ staining of miR-210 (red), its target proteins ISCU1/2 (grey, pointed out by red arrows), and the endothelial marker CD31 (green), miR-210 expression in the KO partners of KO-WT pairs was detected specifically in pulmonary arteriolar endothelium (yellow, micrograph inset), accompanied by a reciprocal decrease in ISCU1/2 expression in these miR-210-positive cells, as compared with KO-KO pairs (N=6 mice/group, average of N=10–12 vessels/mice. Scale bar denotes 50 μm.). (F-H) In parallel, there was no significant difference of endothelin-1 content in plasma (F, N=4 mice/group), nitrate and nitrite content in lungs (G, N=5 mice/group) or inflammatory cytokine IL-6 content in plasma (H, N=4 mice/group) between KO partners of KO-WT pairs with KO-KO pair. (Data are presented as mean ± SEM. In x-axes of (B,D,E,F,G,H) bold font denotes parabionts being studied, whereas regular font denotes partner parabionts. Mann Whitney U test for C,F,G,H and one-way ANOVA with post-hoc Bonferroni testing were performed for other panels.)