Figure 6. PAEC delivery and pathogenic activity of blood-borne miR-210 depends upon extracellular endocrine, rather than myeloid-based or lymphoid-based paracrine, delivery.

(A-B) Mouse parabiosis pairs as in Figure 5 were exposed to chronic hypoxia (10% O₂) for 3 weeks to induce endogenous miR-210 up-regulation. Levels of miR-210 in plasma (A, N=8,7,6,5 mice) and CD31+/CD45− lung endothelial cells (B, N=11,11,10,8 mice) were assessed by RT-qPCR. In KO partners of KO-(WT; Cx3cr1 KO) pairs and KO-(WT; Rag1 KO pairs), they displayed similar extracellular (A) and intracellular (B) miR-210 levels as in KO partners of KO-WT pairs, which was substantially higher than expression in KO-KO pairs. (C) 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), comparable level of miR-210 delivery to pulmonary arteriolar endothelium (yellow, micrograph inset) and corresponding decrease of ISCU1/2 in the KO partners of KO-(WT; Cx3cr1 KO) pairs and of KO-(WT; Rag1 KO) pairs were detected as compared with KO partners of KO-WT pairs (N=6,6,6,5 mice, average of 10–12 vessels/mice. Scale bar denotes 50 μm.). (D-F) In the aspect of pathogenic phenotypes, right ventricular systolic pressure (RVSP) (D, N=7,9,10,6 mice), right ventricular remodeling (E, N=12,8,13,10 mice) and pulmonary arteriolar remodeling and muscularization (F, N=6,6,6,5 mice, average of 10–12 vessels/mice. Scale bar denotes 50 μm.) also showed comparable degree of elevation in KO partners of KO-(WT; Cx3cr1 KO) pairs and of KO-(WT; Rag1 KO) pairs as KO partners of KO-WT pairs, measured by right heart catheterization, RV mass to body weight ratio and in situ staining of α-SMA (white), respectively. (Data are presented as mean ± SEM. In all panels, bold font in x-axes denotes parabionts being studied, whereas regular font denotes partner parabionts. One-way ANOVA with post-hoc Bonferroni testing was performed.)