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Inflammation triggers pulmonary vascular remodelling. Ferroptosis, a nonapoptotic form of cell death that is triggered by iron-dependent lipid peroxidation and contributes to the pathogenesis of several inflammation-related diseases, but its role in pulmonary hypertension (PH) has not been studied. We examined endothelial cell ferroptosis in PH and the potential mechanisms. Pulmonary artery endothelial cells (PAECs) and lung tissues from monocrotaline (MCT)-induced PH rats were analysed for ferroptosis markers, including lipid peroxidation, the labile iron pool (LIP) and the protein expression of glutathione peroxidase 4 (GPX4), ferritin heavy chain 1 (FTH1) and NADPH oxidase-4 (NOX4). The effects of the ferroptosis inhibitor ferrostatin-1 (Fer-1) on endothelial cell ferroptosis and pulmonary vascular remodelling in MCT-induced rats were studied in vitro and in vivo. Ferroptosis was observed in PAECs from MCT-induced PH rats in vitro and in vivo and was characterized by a decline in cell viability accompanied by increases in the LIP and lipid peroxidation, the downregulation of GPX4 and FTH1 expression and the upregulation of NOX4 expression. High-mobility group box 1 (HMGB1)/Toll-like receptor 4 (TLR4)/NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome signalling was measured by western blotting. These changes were significantly blocked by Fer-1 administration in vitro and in vivo. These results suggest that Fer-1 plays a role in inhibiting ferroptosis-mediated PAEC loss during the progression of PH. The ferroptosis-induced inflammatory response depended on the activation of HMGB1/TLR4 signalling, which activated the NLRP3 inflammasome in vivo. We are the first to suggest that pulmonary artery endothelial ferroptosis triggers inflammatory responses via the HMGB1/TLR4/NLRP3 inflammasome signalling pathway in MCT-induced rats. Treating PH with a ferroptosis inhibitor and exploring new treatments based on ferroptosis regulation might be promising therapeutic strategies for PH.

Pulmonary hypertension (PH) is a life-threatening cardiopulmonary disorder whose underlying pathogenesis is unknown. Our previous study showed that pulmonary endothelial cell (PAEC) ferroptosis is involved in the progression of PH by releasing High-mobility group box 1 (HMGB1) and activating Toll-like receptor 4/NOD-like receptor family pyrin domain containing 3 (TLR4/NLRP3) inflammasome signalling. The precise mechanisms that regulate ferroptosis in PH are unclear. This study aimed to investigate the effect of peroxiredoxin 6 (PRDX6) on PAEC ferroptosis in PH.A rat model of PH was established with monocrotaline (MCT), and the distribution and expression of PRDX6 in the pulmonary artery were examined. Lentiviral vectors carrying PRDX6 (LV-PRDX6) were transfected into PAECs and injected into MCT-induced PH rats. Cell viability, MDA levels, reactive oxygen species (ROS) levels, labile iron pool (LIP) levels and mitochondrial morphology were examined. Ferroptosis-related proteins (NADPH oxidase-4 (NOX4), glutathione peroxidase 4 (GPX4), and ferritin heavy chain 1(FTH1)), TLR4, NLRP3 inflammasome markers, HMGB1 and inflammatory cytokines were examined. Pulmonary vascular remodelling and right ventricular structure and function were measured.PRDX6 was expressed in PAECs and was significantly decreased in PH. PRDX6 overexpression significantly inhibited ferroptosis in PAECs under PH conditions in vitro and in vivo, as indicated by increased cell viability, decreased MDA, ROS and LIP levels, inhibited mitochondrial damage, upregulated GPX4 and FTH1 expression, and downregulated NOX4 expression. PRDX6 overexpression attenuated pulmonary vascular remodelling and changes in right ventricle structure and function in MCT-induced PH rats. Moreover, PRDX6 overexpression prevented HMGB1 release by PAECs and decreased TLR4 and NLRP3 inflammasome expression and inflammatory cytokine release in macrophages, while RSL3, a specific activator of ferroptosis, reversed these effects.Taken together, these findings indicate that PRDX6 regulates PAEC ferroptosis through the release of HMGB1 and activation of the TLR4/NLRP3 inflammasome signalling pathway, providing novel therapeutic targets for the treatment of PH.

Introductionambrisentan and phosphodiesterase type 5 inhibitor (PDE5i) have been approved for treating patients with pulmonary arterial hypertension (PAH). Echocardiographic right ventricular pulmonary artery coupling (RVPAC) has been shown to be a valid non-invasive and alternative measurement method to assess the predicted outcomes in PAH patients. The aim of this study was to study the effect and clinical correlates of initial ambrisentan plus PDE5i combination therapy on RVPAC in patients with severe PAH.Method and ResultsWe retrospectively studied and analyzed comprehensive clinical data, hemodynamics, and echocardiography in 27 patients with severe PAH before and after 6 months of initial combination therapy. Compared with the baseline, significant improvements in RVPAC ratios were observed, including RVFAC/PASP (0.31 ± 0.10 vs. 0.44 ± 0.15%/mmHg, p < 0.001), TAPSE/PASP (0.15 ± 0.05 vs. 0.21 ± 0.06 mm/mmHg, p = 0.001), S’/PASP (0.10 ± 0.03 vs. 0.14 ± 0.05 cm/s∙mmHg, p = 0.001), and RVSV/RVESV (0.79 ± 0.22 vs. 1.02 ± 0.20, p < 0.001). Functional status indices [World Health Organization functional classifications (WHO-FC) and 6 min walk distance (6MWD) and N-terminal pro B-type natriuretic peptide (NT-proBNP) levels] showed significant improvements. Right heart catheterization (RHC) evaluations for hemodynamic measurements between baseline and the 6–12 month follow-up were sPAP (96 ± 22 vs. 86 ± 24 mmHg, p = 0.002), mPAP (64 ± 18 vs. 56 ± 17 mmHg, p < 0.001) and TPVR (17.3 ± 6.7 vs. 12.1 ± 5.4 WU, p = 0.001). Simultaneously, significant associations between RVPAC ratios and NT-proBNP levels and WHO-FC and 6MWD were observed.ConclusionAmbrisentan plus PDE-5i combination therapy resulted in a significant improvement in RVPAC in severe PAH. Importantly, RVPAC parameters correlated with known prognostic markers of PAH.