MicroRNA-483 amelioration of experimental pulmonary hypertension

Abstract

Endothelial dysfunction is critically involved in the pathogenesis of pulmonary arterial hypertension (PAH) and that exogenously administered microRNA may be of therapeutic benefit. Lower levels of miR-483 were found in serum from patients with idiopathic pulmonary arterial hypertension (IPAH), particularly those with more severe disease. RNA-seq and bioinformatics analyses showed that miR-483 targets several PAH-related genes, including transforming growth factor-β (TGF-β), TGF-β receptor 2 (TGFBR2), β-catenin, connective tissue growth factor (CTGF), interleukin-1β (IL-1β), and endothelin-1 (ET-1). Overexpression of miR-483 in ECs inhibited inflammatory and fibrogenic responses, revealed by the decreased expression of TGF-β, TGFBR2, β-catenin, CTGF, IL-1β, and ET-1. In contrast, inhibition of miR-483 increased these genes in ECs. Rats with EC-specific miR-483 overexpression exhibited ameliorated pulmonary hypertension (PH) and reduced right ventricular hypertrophy on challenge with monocrotaline (MCT) or Sugen + hypoxia. A reversal effect was observed in rats that received MCT with inhaled lentivirus overexpressing miR-483. These results indicate that PAH is associated with a reduced level of miR-483 and that miR-483 might reduce experimental PH by inhibition of multiple adverse responses.

Keywords: TGF-β; endothelium; miR-483; pulmonary hypertension.

Figure 1. Lower serum miR‐483 level in IPAH patients

1. A

   Serum levels of miR‐483‐3p/‐5p in IPAH patients (n = 139) and HC (n = 95) measured by qPCR. The data are fold change normalized to the averaged level of HC.

2. B

   ROC curve with sensitivity and specificity of serum levels of miR‐483‐3p/‐5p for differentiating IPAH patients from HCs at diagnosis (IPAH, n = 139; HC, n = 95).

3. C

   Levels of miR‐483‐3p/‐5p associated with PAH risk in three groups. IPAH patients were divided into a low‐risk group (Low) and an intermediate‐ plus high‐risk group (Inter&high) according to the World Symposium on Pulmonary Hypertension 2018 [14].

4. D

   Levels of miR‐483‐3p were inversely correlated with serum levels of ET‐1 (IPAH, n = 118; HC, n = 95) and IL‐6 (IPAH, n = 112; HC, n = 93).

Data information: Values are expressed as median ± interquartile range. Statistical test: Mann–Whitney U‐test (A), Kruskal–Wallis U‐test (C).Source data are available online for this figure.

Figure 2. MiR‐483 targets PAH‐related genes and pathways in PAECs

1. A

   CD144‐enriched EVs were isolated from serum of IPAH patients (n = 37) and HCs (n = 34). The levels of miR‐483‐3p/‐5p were measured by qPCR.

2. B, C

   PAECs were transfected with miR‐483‐3p/‐5p mimic or scramble RNA for 36 hr before RNA isolation and then analyzed by RNA‐seq. Data are results from two biological repeats. (B) PAH‐related GO enrichment delineated by DAVID for the top 300 upregulated or downregulated genes with the cutoff of P < 0.05. (C) Heat map comparison of log2 fold changes of the indicated genes.

Data information: Values are expressed as median ± interquartile range. Statistical test: Mann–Whitney U‐test.Source data are available online for this figure.

Figure 3. MiR‐483‐targeted genes

1. A

   Predicted binding sites for miR‐483‐3p/‐5p on the 3′UTR of mRNAs as indicated.

2. B–D

   PAECs were infected with Lenti‐pre‐miR‐483 or Lenti‐null for 24 hr. Expression levels of miR‐483‐3p/‐5p, TGF‐β, TGFBR2, β‐catenin, CTGF, IL‐1β, and ET‐1 mRNA and protein were measured by qPCR and Western blot, respectively.

3. E

   Bovine aortic ECs were transfected with a luciferase reporter fused with the 3′UTR of TGF‐β (Luc‐TGF‐β WT), TGFBR2 (Luc‐TGFBR2 WT), IL‐1β (Luc‐IL‐1β WT), or ET‐1 (Luc‐ET‐1 WT) or a binding site mutation (Luc‐TGF‐β Mut, Luc‐TGFBR2 Mut, Luc‐IL‐1β Mut, ET‐1‐Mut), then infected with Lenti‐pre‐miR‐483 for additional 36 hr. Luciferase activity was measured.

4. F, G

   PAECs were infected with Lenti‐pre‐miR‐483 or Lenti‐null for 36 h. The Ago1‐ or Ago2‐associated miRNAs and mRNAs were enriched by immunoprecipitation with anti‐Ago1 or anti‐Ago2. Levels of miR‐483‐3p/‐5p and TGF‐β, TGFBR2, β‐catenin, CTGF, IL‐1β, and ET‐1 mRNA were detected by qPCR and normalized to those of Ago1 or Ago2 protein.

Data information: Values are expressed as mean ± SEM from three independent experiments. Statistical test: t‐test (*P < 0.05 between the indicated groups, exact P‐values are shown in Appendix Table S3).Source data are available online for this figure.

Figure 4. Decreased level of PAH‐induced genes in EC‐miR‐483 Tg rats

1. A

   Lung ECs were isolated from EC‐miR‐483 transgenic rats (Tg) and their wild‐type littermates (WT), n = 3 in each group. MiR‐483‐3p/‐5p levels were measured by qPCR.

2. B

   Serum from WT and Tg rats (n = 6) was collected. MiR‐483‐3p and miR‐483‐5p associated with CD144 were enriched by immunoprecipitation with anti‐CD144. Levels of miR‐483‐3p/‐5p were measured by qPCR.

3. C, D

   Lung ECs isolated from miR‐483‐Tg rat and WT littermates were exposed to normoxia or hypoxia (0.2% O₂) for 24 hr. Proliferation and migration of ECs were measured by flow cytometry and wound healing assay, respectively.

4. E–H

   Tg and WT rats were injected with saline or MCT. Expression levels of TGF‐β, TGFBR2, β‐catenin, CTGF, IL‐1β, and ET‐1 mRNA and protein in lung tissues were measured by qPCR and Western blot, respectively (E). Levels of miR‐483‐3p/‐5p in serum and lung tissues were measured by qPCR (F). Ago1‐ or Ago2‐associated miRNAs (G) and mRNAs (H) were enriched from the isolated lung tissues by immunoprecipitation with anti‐Ago1 or anti‐Ago2 and quantified by qPCR (n = 3×3, samples were pooled from three animals for each assay, and 3 independent experiments [a total of 9 animals] were performed).

Data information: Values are expressed as mean ± SEM. Data are representative of three independent experiments (C, D). Statistical test: t‐test (*P < 0.05 vs. respective controls or between the indicated groups, exact P‐values are shown in Appendix Table S3).Source data are available online for this figure.

Figure 5. EC‐miR‐483 Tg rats show ameliorated PH

1. A–D

   EC‐miR‐483‐Tg and WT rats were injected with saline or MCT. Analysis of mPAP and RV hypertrophy [RV/(LV + S)] for the indicated groups (7 rats per group) (A, B). Vascularization evaluated by H&E staining of pulmonary arteries (C). Vascular integrity evaluated by intravenous injection of Evans blue dye (D).

2. E, F

   EC‐miR‐483‐Tg rats and WT littermates were injected with SU5416 and then exposed to hypoxia for 3 weeks and had reoxygenation for 2 weeks or injected with DMSO and exposed to normoxia for 5 weeks. Analysis of mPAP (E) and RV hypertrophy [RV/(LV + S)] (F) for the indicated groups.

Data information: Values are expressed as mean ± SEM. Statistical test: t‐test (*P < 0.05 vs. respective controls or between the indicated groups). Scale bar: 20 μm (C) or 0.5 cm (D).Source data are available online for this figure.

Figure 6. Lenti‐miR‐483 inhalation ameliorates MCT‐induced PAH

1. A

   Ctrl‐ or MCT‐treated rats were intratracheally delivered Lenti‐pre‐miR‐483‐GFP or Lenti‐GFP at Day 21 and Day 28 after the start of MCT treatment (S+GFP, M+GFP, and M+483).

2. B, C

   qPCR analysis of miR‐483‐3p/‐5p in serum and lung tissue from PH (MCT‐treated) rats.

3. D

   Protein and mRNA levels of TGF‐β, TGFBR2, β‐catenin, CTGF, IL‐1β, and ET‐1 measured by Western blot and qPCR, respectively (n = 3×3, samples were pooled from three animals for each assay, and 3 independent experiments [a total of 9 animals] were performed).

4. E–I

   Analysis of mPAP (E), RV hypertrophy [RV/(LV + S)] (F), and systemic blood pressure (SBP) (G) for the indicated groups (7 rats in each group). (H) RV hypertrophy shown by the H&E staining and RV/(LV + S) quantification. (I) Vascularization revealed by H&E staining (n = 7 rats in each group).

5. J

   Survival rate of three groups (n = 15 rats in each group).

Data information: Values are expressed as mean ± SEM. Statistical test: t‐test (*P < 0.05 vs. respective controls or between the indicated groups, exact P‐values are shown in Appendix Table S3). Scale bar: 0.5 cm (H) or 20 μm (I).Source data are available online for this figure.