Aerosolized gene and oligonucleotide therapy targeting microRNA-224 ameliorates pulmonary hypertension and cor pulmonale by orchestrating the BMP and TGFß signaling

Abstract

Pulmonary hypertension (PH) is a progressive pulmonary vascular disease that leads to right heart failure and ultimately death. Despite intensive research in the last decades effective root-cause therapies remain elusive. MicroRNAs (miRs) have emerged as critical regulators of cardiovascular homeostasis and disease. Here, we pinpoint microRNA-224-5p (miR-224) as a key regulator of pulmonary vascular remodeling and delineate its mechanism of action in PAH. Moreover, we use both a cell-specific gene therapeutic and an oligonucleotide-based approach to treat PH in three distinct animal rodent models.

MiR-224 expression was quantified in PH lungs and pulmonary vascular cells across species using qPCR. Smooth muscle cells from healthy individuals and PH patients were isolated, and their proliferation upon microRNA-224 stimulation was assessed. We performed in vivo studies in three distinct animal models.

In vivo, we first overexpressed miR-224 using intra-tracheal aerosolization in the mouse sugen/hypoxia model. Then, using the same animal model, we inhibited microRNA-224 using AAV1-Tough-Decoy-miR-224 or an oligonucleotide approach (locked nucleic acids, LNA). Additionally, we developed a smooth muscle cell-specific AAV and inhibited microRNA-224 through intra-tracheal aerosolisation in PH-diseased mice. In a translational approach, we used intra-tracheally aerosolised LNAs and therapeutically inhibited miR-224 in the rat models of sugen/hypoxia and monocrotaline (MCT). Functional assessment was performed using right heart catheterization. Furthermore, to assess right heart function, we performed MRI in LNA-treated MCT rats.

We performed single-cell RNA sequencing on lungs from normoxic or Sugen/Hypoxia mice treated with LNA-Ctrl or LNA-224. Targets identified were verified using qPCR, immunoblots, and mutagenesis assays.

MiR-224 expression was markedly increased cross species in PH. In vitro, miR-224 overexpression was sufficient to induce PASMC proliferation. In vivo miR-224 exacerbated PAH in mice, whereas intratracheal delivery of aerosolized AAV1-ToughDecoy-miR-224 or a chemically modified antisense oligonucleotide targeting miR-224 (LNA-224) significantly attenuated disease severity in Sugen/Hypoxia and monocrotaline models.

Invasive right heart catheterisation revealed a significant reduction of pulmonary artery and right ventricular pressure. Furthermore, right heart function, assessed non invasively in the MRI studies was markedly ameliorated.

Moreover, SMC inhibition of miR-224 using a smooth muscle cell specific AAV reversed pulmonary vascular remodeling. Mechanistically, miR-224 directly targets multiple components of the BMP and TGF-β signaling pathways, leading to suppression of BMP/SMAD signaling and enhancement of TGF-β-associated responses.

MiR-224 is a central regulator of pulmonary vascular remodeling and cor pulmonale. Aerosolized gene and oligonucleotide therapy shows therapeutic promise for PH.