Reversal of MicroRNA Dysregulation in an Animal Model of Pulmonary Hypertension

Abstract

Background: Animals models have played an important role in enhancing our understanding of the pathogenesis of pulmonary arterial hypertension (PAH). Dysregulation of the profile of microRNAs (miRNAs) has been demonstrated in human tissues from PAH patients and in animal models. In this study, we measured miRNA levels in the monocrotaline (MCT) rat model of PAH and examined whether blocking a specific dysregulated miRNA not previously reported in this model, attenuated PAH. We also evaluated changes in miRNA expression in lung specimens from MCT PAH rats overexpressing human prostacyclin synthase, which has been shown to attenuate MCT PAH.

Methods: Expression levels of a panel of miRNAs were measured in MCT-PAH rats as compared to naïve (saline) control rats. Subsequently, MCT PAH rats were injected with a specific inhibitor (antagomiR) for miR-223 (A223) or a nonspecific control oligonucleotide (A-control) 4 days after MCT administration, then weekly. Three weeks later, RV systolic pressure and RV mass were measured. Total RNA, isolated from the lungs, microdissected pulmonary arteries, and right ventricle, was reverse transcribed and real-time quantitative PCR was performed. MiRNA levels were also measured in RNA isolated from paraffin sections of MCT-PAH rats overexpressing prostacyclin synthase.

Results: MiRs 17, 21, and 223 were consistently upregulated, whereas miRs 126, 145, 150, 204, 424, and 503 were downregulated in MCT PAH as compared to vehicle control. A223 significantly reduced levels of miR-223 in PA and lungs of MCT PAH rats as compared to levels measured in A-control or control MCT PAH rats, but A223 did not attenuate MCT PAH. Right ventricular mass and right ventricular systolic pressure in rats treated with A223 were not different from values in A-control or MCT PAH rats. In contrast, analysis of total RNA from lung specimens of MCT PAH rats overexpressing human prostacyclin synthase (hPGIS) demonstrated reversal of MCT-induced upregulation of miRs 17, 21, and 223 and an increase in levels of miR-424 and miR-503. Reduction in bone morphogenetic receptor 2 (BMPR2) messenger (m)RNA expression was not altered by A223, whereas human prostacyclin synthase overexpression restored BMPR2 mRNA to levels in MCT PAH to levels measured in naive controls.

Conclusions: Inhibition of miR-223 did not attenuate MCT PAH, whereas human prostacyclin synthase overexpression restored miRNA levels in MCT PAH to levels detected in naïve rats. These data may establish a paradigm linking attenuation of PAH to restoration of BMPR2 signaling.

Fig 1. MiRNA expression in MCT PAH rats.

Expression levels were measured 21–24 days after MCT treatment and are relative to saline-saline (naïve) control animals. (A) Lungs. (B) PA. (C) RV. (D). Plasma. *Significantly different from saline-saline control; n = 8. --- Saline-saline control reference line.

Fig 2. Comparison of normalization methods in plasma.

MiR-103 and c. elegans miR-39 normalization methods were compared in plasma from MCT PAH rats. N = 4. p values are as follows: MiR-126 p = 0.499, miR-145 p = 0.422, miR-150 p = 0.803.

Fig 3. Effect of A223 administration on miRNA expression in MCT PAH.

Expression levels of miR-223 relative to saline-saline (naïve) control animals. (A) Lungs. (B) PA. (C) RV. (D) Plasma.—Saline-saline control reference line. (E) Expression of miRNAs in the PA of MCT-A223 animals relative to MCT-A-control animals.—MCT-A- control reference line.*Significantly different from saline-saline control; ^†Significantly different from MCT-saline and MCT-A- control; n = 8.

Fig 4. Functional assessment of A223 treatment in rats after MCT administration.

(A) RV/(LV+S) ratios, n = 8. (B) RVSP measurements, n = 4–7. *Significantly different from saline-saline control.

Fig 5. Assessment of AAV-mediated overexpression of hPGIS on miRNA expression in the formalin-fixed paraffin-embedded lung specimens from MCT PAH.

Functional assessment of AAV-mediated overexpression of hPGIS in MCT PAH rats. (A) RV/(LV+S) ratios. (B) RVSP measurements. (C) MiRNA expression in lung specimens from MCT-saline and MCT-AAV-hPGIS rats relative to saline-saline (naïve) control; *Significantly different from saline-saline control; ^†Significantly different from MCT-saline control; n = 4.—Saline-saline control reference line. Hemodynamic data is adapted from reference [28].

Fig 6. Changes in BMPR2 and IGF-1R mRNA expression in MCT PAH.

(A) Expression in lungs and PA after A223 administration, n = 8. (B) Expression in lungs after hPGIS overexpression mediated attenuation of MCT PAH, n = 4. *Significantly different from saline-saline control; ^†Significantly different from MCT-saline control.—Saline-saline control reference line.