Long Range Endocrine Delivery of Circulating miR-210 to Endothelium Promotes Pulmonary Hypertension

Abstract

Rationale: Unproven theories abound regarding the long-range uptake and endocrine activity of extracellular blood-borne microRNAs into tissue. In pulmonary hypertension (PH), microRNA-210 (miR-210) in pulmonary endothelial cells promotes disease, but its activity as an extracellular molecule is incompletely defined.

Objective: We investigated whether chronic and endogenous endocrine delivery of extracellular miR-210 to pulmonary vascular endothelial cells promotes PH.

Methods and results: Using miR-210 replete (wild-type [WT]) and knockout mice, we tracked blood-borne miR-210 using bone marrow transplantation and parabiosis (conjoining of circulatory systems). With bone marrow transplantation, circulating miR-210 was derived predominantly from bone marrow. Via parabiosis during chronic hypoxia to induce miR-210 production and PH, miR-210 was undetectable in knockout-knockout mice pairs. However, in plasma and lung endothelium, but not smooth muscle or adventitia, miR-210 was observed in knockout mice of WT-knockout pairs. This was accompanied by downregulation of miR-210 targets ISCU (iron-sulfur assembly proteins)1/2 and COX10 (cytochrome c oxidase assembly protein-10), indicating endothelial import of functional miR-210. Via hemodynamic and histological indices, knockout-knockout pairs were protected from PH, whereas knockout mice in WT-knockout pairs developed PH. In particular, pulmonary vascular engraftment of miR-210-positive interstitial lung macrophages was observed in knockout mice of WT-knockout pairs. To address whether engrafted miR-210-positive myeloid or lymphoid cells contribute to paracrine miR-210 delivery, we studied miR-210 knockout mice parabiosed with miR-210 WT; Cx3cr1 knockout mice (deficient in myeloid recruitment) or miR-210 WT; Rag1 knockout mice (deficient in lymphocytes). In both pairs, miR-210 knockout mice still displayed miR-210 delivery and PH, thus demonstrating a pathogenic endocrine delivery of extracellular miR-210.

Conclusions: Endogenous blood-borne transport of miR-210 into pulmonary vascular endothelial cells promotes PH, offering fundamental insight into the systemic physiology of microRNA activity. These results also describe a platform for RNA-mediated crosstalk in PH, providing an impetus for developing blood-based miR-210 technologies for diagnosis and therapy in this disease.

Keywords: endothelium; lymphocytes; macrophage; microRNAs; parabiosis.

Figure 1.. A majority of circulating, steady-state hypoxia-induced miR-210 is derived from bone marrow cells.

(A) At 12 weeks post-bone marrow transplantation of miR-210 (WT) and miR-210 (KO) mice with WT or KO bone marrow, mice were exposed to chronic hypoxia (10% O₂) for 3 weeks to induce miR-210 up-regulation. (B-C) By RT-qPCR, circulating extracellular miR-210 (B, N=5,6,6,6 mice) and intracellular miR-210 (C, N=4,4,5,4 mice) were detected and quantified in plasma (B) and CD31+/CD45− lung endothelial cells (C) of WT mice and KO mice transplanted with WT or KO bone marrow. (One-way ANOVA with post-hoc Bonferroni testing was performed.)

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.

(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.)

Figure 3.. Delivery of blood-borne miR-210 promotes PH.

(A) By invasive right heart catheterization and measurement of right ventricular systolic pressure (RVSP), while hypoxic KO-KO pairs were protected from PH as compared with WT-WT pairs, the KO partners in KO-WT pairs developed significantly higher RVSP versus KO-KO pairs (N=7,9,6,8 mice). (B) Right ventricular mass/body weight ratio was increased in the KO partners of KO-WT pairs as compared with KO-KO pairs (N=12,8,7,9 mice). (C) In situ staining of α-SMA (green) demonstrated increased pulmonary arteriolar remodeling and muscularization in the KO partners of KO-WT pairs versus KO-KO pairs and comparable to that seen in WT partners and WT-WT pairs (N=6 mice/group, average of 10–12 vessels/mice. Scale bar denotes 50 μm.). (Data are presented as mean ± SEM. In x-axes of (A,B,C), bold font denotes parabionts being studied, whereas regular font denotes partner parabionts. One-way ANOVA with post-hoc Bonferroni testing was performed.)

Figure 4.. Hypoxic expansion of miR-210-positive blood monocytes and interstitial lung macrophages.

(A) Employing the same parabiosis system as in Figure 2 and via fluorescence activated cell sorting (FACS) of blood, total blood monocytes (N=6,6,5,6 mice), and subpopulations including Ly6c^high and Ly6c^low monocytes (N=5,4,4,4 mice) were increased in hypoxic WT-WT pairs as compared with hypoxic KO-KO pairs. Importantly, KO mice of KO-WT pairs displayed an equivalent level of total blood monocytes and Ly6c^high monocytes, which was significantly higher than KO-KO pairs. (B-C) In parallel, by FACS, expansion of CD64/CD11b-positive interstitial lung macrophages was observed in isolated cells from whole lung of KO mice of KO-WT pairs comparable to that seen in the WT partner and WT-WT pairs but substantially increased compared with KO-KO pairs (quantitation in B (N=6,6,5,6 mice, and representative flow cytometric plots in C. Percentages reflect the frequencies of interstitial macrophages among CD45.2+ Siglec F- Ly-6G- CD11b+ MHC class II- leukocytes in the lung.). (D) Increased presence of miR-210-positive interstitial lung macrophages (yellow, micrograph inset) was also demonstrated by in situ staining of CD68 (red) and miR-210 (green) in the small pulmonary arterioles of KO mice of KO-WT pairs, comparable to WT partners and WT-WT pairs but greater in number than those seen in KO-KO pairs (N=6 mice/group, average of 10–12 vessels/mice. Scale bar denotes 50 μm.). (E) By FACS, no significant difference of lymphocyte presence was observed in lung tissue of KO-KO pairs, KO-WT pairs and WT-WT pairs (N=6,6,4,6 mice). (Data are presented as mean ± SEM. In x-axes of (A,B,D,E), bold font denotes parabionts being studied, whereas regular font denotes partner parabionts. One-way ANOVA with post-hoc Bonferroni testing was performed.)

Figure 5.. Transfer of miR-210-positive myeloid cells and lymphocytes was abrogated by Cx3cr1 deficiency and Rag1 deficiency, respectively.

(A) A parabiosis platform similar to that in Figure 2 was used in the context of pairing miR-210 −/− (KO) mice with miR-210 −/− (KO), miR-210 +/+ (WT), miR-210 +/+; Cx3cr1 −/− (WT; Cx3cr1 KO) or miR-210 +/+; Rag1 −/− (WT; Rag1 KO) mice. One week after surgery, the mouse pairs were exposed to chronic hypoxia (10% O₂) for three weeks. (Red arrows denote comparison groups) (B) As denoted by frequencies (%) of enriched lymphocytes within the total leukocyte population, lack of mature lymphocytes was confirmed by flow cytometry in blood and lung tissue of (WT; Rag1 KO mice). (C) Decreased presence of miR-210-positive interstitial lung macrophages (yellow, micrograph inset) was demonstrated by in situ staining of CD68 (red) and miR-210 (green) in the small pulmonary arterioles of KO partners of KO-(WT; Cx3cr1 KO) pairs, comparable to those seen in KO-KO pairs (N=6 mice/group, average of 10–12 vessels/mice. Scale bar denotes 50 μm.). (D) In parallel, circulating levels of inflammatory marker IL6 were not altered in plasma of KO partners of KO-WT pairs, KO-(WT; Cx3cr1 KO) pairs and KO-(WT; Rag1 KO) compared to KO-KO pairs, as quantified by ELISA (N=4 mice/group). (Data are presented as mean ± SEM. In x-axes of (C,D), bold font denotes parabionts being studied, whereas regular font denotes partner parabionts. Kruskal Wallis and post-hoc Dunn’s testing for D and one-way ANOVA with post-hoc Bonferroni testing for other panels were performed.)

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.)

Figure 7.. Model of the endocrine delivery and pathogenic actions of blood-borne miR-210 in PH.

Collectively, our findings demonstrate that long range, endocrine transport encompasses the predominant mechanism by which blood-borne miR-210 is delivered into pulmonary arterial endothelial cells with consequent control of PH in vivo.