Single nuclei RNA-sequencing unveils alveolar macrophages as drivers of endothelial damage in obese HFpEF-related pulmonary hypertension

Background
Pulmonary hypertension (PH) is a frequent complication in obese patients showing heart failure with preserved ejection fraction (HFpEF) and correlates with poor prognosis. PH associated with cardiometabolic HFpEF (PH-cHFpEF) is characterized by inflammation and metabolic dysregulation. Alterations in the immune landscape, particularly activation of alveolar macrophages (AMs), may propagate the inflammatory response and lead to endothelial damage and vascular remodeling in the lung. Whether AMs contribute to PH in cardiometabolic HFpEF remains elusive.

Purpose
The present study investigates the role of alveolar macrophages in PH-cHFpEF.

Methods
Mice subjected to high-fat diet and L-NAME treatment for 15 weeks were used as experimental model of PH-cHFpEF. At the end of the treatment, echocardiography and treadmill exhaustion tests were performed. Single nucleus RNA-sequencing (snRNA-seq) was employed to study the AMs transcriptional landscape and cell-cell interactions. In vitro experiments were performed to study the mechanisms underlying metabolic stress-induced macrophage dysfunction using palmitic acid (PA), co-culture experiments were used to investigate the crosstalk between macrophages and endothelial cells.

Results
Compared with control mice, PH-cHFpEF animals displayed right ventricular dysfunction, vascular remodeling and increased pulmonary pressure. SnRNA-seq of mouse lungs revealed transcriptional alterations in AMs, with a significant reduction in their abundance in PH-cHFpEF mice. These changes were associated with dysregulation of transcriptional programs involved in pyroptosis, defective autophagy and inflammation in AMs from PH-cHFpEF vs. control mice, as shown by the upregulation of c-Fos, Dusp1, Pim-1 and Ccn1. STRING analysis revealed a molecular link between these partners and highlighted c-Fos/Dusp-1 as a central axis of AMs cell death and inflammation. Metabolic stress induced by PA in isolated murine macrophages recapitulated c-Fos/Dusp-1 activation as well as IL-1β, TNF-α, and Caspase-1 upregulation resulting in inflammation, impaired autophagy and enhanced pyroptosis. Moreover, c-Fos/Dusp1 activation in macrophages promoted secretion of pro-inflammatory chemokines leading to endothelial dysfunction in a paracrine manner. Dusp1 knockdown rescued autophagy and pyroptosis while mitigating macrophage-driven inflammation and endothelial damage.

Conclusions
PH-cHFpEF is characterized by AMs activation, upregulation of the cFos/Dusp-1 pathway and subsequent pyroptosis and inflammation in alveolar macrophages. Our findings highlight the role of AMs as putative targets for preventing endothelial damage in experimental PH-cHFpEF.