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Pulmonary arterial hypertension (PAH) is a devastating lung disease characterized by the progressive obstruction of the distal pulmonary arteries (PA). Structural and functional alteration of pulmonary artery smooth muscle cells (PASMC) and endothelial cells (PAEC) contributes to PA wall remodeling and vascular resistance, which may lead to maladaptive right ventricular (RV) failure and, ultimately, death. Here, we found that decreased expression of sarcoplasmic/endoplasmic reticulum Ca 2+ ATPase 2a (SERCA2a) in the lung samples of PAH patients was associated with the down-regulation of bone morphogenetic protein receptor type 2 (BMPR2) and the activation of signal transducer and activator of transcription 3 (STAT3). Our results showed that the antiproliferative properties of SERCA2a are mediated through the STAT3/BMPR2 pathway. At the molecular level, transcriptome analysis of PASMCs co-overexpressing SERCA2a and BMPR2 identified STAT3 amongst the most highly regulated transcription factors. Using a specific siRNA and a potent pharmacological STAT3 inhibitor (STAT3i, HJC0152), we found that SERCA2a potentiated BMPR2 expression by repressing STAT3 activity in PASMCs and PAECs. In vivo, we used a validated and efficient model of severe PAH induced by unilateral left pneumonectomy combined with monocrotaline (PNT/MCT) to further evaluate the therapeutic potential of single and combination therapies using adeno-associated virus (AAV) technology and a STAT3i. We found that intratracheal delivery of AAV1 encoding SERCA2 or BMPR2 alone or STAT3i was sufficient to reduce the mean PA pressure and vascular remodeling while improving RV systolic pressures, RV ejection fraction, and cardiac remodeling. Interestingly, we found that combined therapy of AAV1.hSERCA2a with AAV1.hBMPR2 or STAT3i enhanced the beneficial effects of SERCA2a. Finally, we used cardiac magnetic resonance imaging to measure RV function and found that therapies using AAV1.hSERCA2a alone or combined with STAT3i significantly inhibited RV structural and functional changes in PNT/MCT-induced PAH. In conclusion, our study demonstrated that combination therapies using SERCA2a gene transfer with a STAT3 inhibitor could represent a new promising therapeutic alternative to inhibit PAH and to restore BMPR2 expression by limiting STAT3 activity.

Aims: Cyclic adenosine monophosphate (cAMP) is the predominant intracellular second messenger that transduces signals from Gs-coupled receptors. Intriguingly, there is evidence from various cell types that an extracellular cAMP pathway is active in the extracellular space. Herein, we investigated the role of extracellular cAMP in the lung and examined whether it may act on pulmonary vascular cell proliferation and pulmonary vasculature remodelling in the pathogenesis of pulmonary hypertension (PH)., Methods and Results: The expression of cyclic AMP-metabolizing enzymes was increased in lungs from patients with PH as well as in rats treated with monocrotaline and mice exposed to Sugen/hypoxia. We report that inhibition of the endogenous extracellular cAMP pathway exacerbated Sugen/hypoxia-induced lung remodelling. We found that application of extracellular cAMP induced an increase in intracellular cAMP levels and inhibited proliferation and migration of pulmonary vascular cells in vitro. Extracellular cAMP infusion in two in vivo PH models prevented and reversed pulmonary and cardiac remodelling associated with PH. Using protein expression analysis along with luciferase assays, we found that extracellular cAMP acts via the A2R/PKA/CREB/p53/Cyclin D1 pathway., Conclusions: Taken together, our data reveal the presence of an extracellular cAMP pathway in pulmonary arteries that attempts to protect the lung during PH, and suggest targeting of the extracellular cAMP signalling pathway to limit pulmonary vascular remodelling and PH., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2019. For permissions, please email: journals.permissions@oup.com.)

Background: Pulmonary arterial hypertension (PAH) results in right ventricular (RV) failure, electro-mechanical dysfunction and heightened risk of sudden cardiac death (SCD), although exact mechanisms and predisposing factors remain unclear. Because impaired chronotropic response to exercise is a strong predictor of early mortality in patients with PAH, we hypothesized that progressive elevation in heart rate can unmask ventricular tachyarrhythmias (VT) in a rodent model of monocrotaline (MCT)-induced PAH. We further hypothesized that intra-tracheal gene delivery of aerosolized AAV1.SERCA2a (AAV1.S2a), an approach which improves pulmonary vascular remodeling in PAH, can suppress VT in this model., Objective: To determine the efficacy of pulmonary AAV1.S2a in reversing electrophysiological (EP) remodeling and suppressing VT in PAH., Methods: Male rats received subcutaneous injection of MCT (60 mg/kg) leading to advanced PAH. Three weeks following MCT, rats underwent intra-tracheal delivery of aerosolized AAV1.S2a (MCT + S2a, N = 8) or saline (MCT, N = 9). Age-matched rats served as controls (CTRL, N = 7). The EP substrate and risk of VT were determined using high-resolution optical action potential (AP) mapping ex vivo. The expression levels of key ion channel subunits, fibrosis markers and hypertrophy indices were measured by RT-PCR and histochemical analyses., Results: Over 80% of MCT but none of the CTRL hearts were prone to sustained VT by rapid pacing (P < .01). Aerosolized gene delivery of AAV1.S2a to the lung suppressed the incidence of VT to <15% (P < .05). Investigation of the EP substrate revealed marked prolongation of AP duration (APD), increased APD heterogeneity, a reversal in the trans-epicardial APD gradient, and marked conduction slowing in untreated MCT compared to CTRL hearts. These myocardial EP changes coincided with major remodeling in the expression of K and Ca channel subunits, decreased expression of Cx43 and increased expression of pro-fibrotic and pro-hypertrophic markers. Intra-tracheal gene delivery of aerosolized AAV1 carrying S2a but not luciferase resulted in selective upregulation of the human isoform of SERCA2a in the lung but not the heart. This pulmonary intervention, in turn, ameliorated MCT-induced APD prolongation, reversed spatial APD heterogeneity, normalized myocardial conduction, and suppressed the incidence of pacing-induced VT. Comparison of the minimal conduction velocity (CV) generated at the fastest pacing rate before onset of VT or at the end of the protocol revealed significantly lower values in untreated compared to AAV1.S2a treated PAH and CTRL hearts. Reversal of EP remodeling by pulmonary AAV1.S2a gene delivery was accompanied by restored expression of key ion channel transcripts. Restored expression of Cx43 and collagen but not the pore-forming Na channel subunit Nav1.5 likely ameliorated VT by improving CV at rapid rates in PAH., Conclusion: Aerosolized AAV1.S2a gene delivery selectively to the lungs ameliorates myocardial EP remodeling and VT susceptibility at rapid heart rates. Our findings highlight for the first time the utility of a non-cardiac gene therapy approach for arrhythmia suppression., (Copyright © 2018 Elsevier Ltd. All rights reserved.)