Relationship between impaired BMP signalling and clinical risk factors at early-stage vascular injury in the preterm infant.

INTRODUCTION: Chronic lung disease, that is, bronchopulmonary dysplasia (BPD) is the most common complication in preterm infants and develops as a consequence of the misguided formation of the gas-exchange area undergoing prenatal and postnatal injury. Subsequent vascular disease and its progression into pulmonary arterial hypertension critically determines long-term outcome in the BPD infant but lacks identification of early, disease-defining changes. METHODS: We link impaired bone morphogenetic protein (BMP) signalling to the earliest onset of vascular pathology in the human preterm lung and delineate the specific effects of the most prevalent prenatal and postnatal clinical risk factors for lung injury mimicking clinically relevant conditions in a multilayered animal model using wild-type and transgenic neonatal mice. RESULTS: We demonstrate (1) the significant reduction in BMP receptor 2 (BMPR2) expression at the onset of vascular pathology in the lung of preterm infants, later mirrored by reduced plasma BMP protein levels in infants with developing BPD, (2) the rapid impairment (and persistent change) of BMPR2 signalling on postnatal exposure to hyperoxia and mechanical ventilation, aggravated by prenatal cigarette smoke in a preclinical mouse model and (3) a link to defective alveolar septation and matrix remodelling through platelet derived growth factor-receptor alpha deficiency. In a treatment approach, we partially reversed vascular pathology by BMPR2-targeted treatment with FK506 in vitro and in vivo. CONCLUSION: We identified impaired BMP signalling as a hallmark of early vascular disease in the injured neonatal lung while outlining its promising potential as a future biomarker or therapeutic target in this growing, high-risk patient population.

Single-cell RNA sequencing profiling of mouse endothelial cells in response to pulmonary arterial hypertension.

AIMS: Endothelial cell (EC) dysfunction drives the initiation and pathogenesis of pulmonary arterial hypertension (PAH). We aimed to characterize EC dynamics in PAH at single-cell resolution. METHODS AND RESULTS: We carried out single-cell RNA sequencing (scRNA-seq) of lung ECs isolated from an EC lineage-tracing mouse model in Control and SU5416/hypoxia-induced PAH conditions. EC populations corresponding to distinct lung vessel types, including two discrete capillary populations, were identified in both Control and PAH mice. Differential gene expression analysis revealed global PAH-induced EC changes that were confirmed by bulk RNA-seq. This included upregulation of the major histocompatibility complex class II pathway, supporting a role for ECs in the inflammatory response in PAH. We also identified a PAH response specific to the second capillary EC population including upregulation of genes involved in cell death, cell motility, and angiogenesis. Interestingly, four genes with genetic variants associated with PAH were dysregulated in mouse ECs in PAH. To compare relevance across PAH models and species, we performed a detailed analysis of EC heterogeneity and response to PAH in rats and humans through whole-lung PAH scRNA-seq datasets, revealing that 51% of up-regulated mouse genes were also up-regulated in rat or human PAH. We identified promising new candidates to target endothelial dysfunction including CD74, the knockdown of which regulates EC proliferation and barrier integrity in vitro. Finally, with an in silico cell ordering approach, we identified zonation-dependent changes across the arteriovenous axis in mouse PAH and showed upregulation of the Serine/threonine-protein kinase Sgk1 at the junction between the macro- and microvasculature. CONCLUSION: This study uncovers PAH-induced EC transcriptomic changes at a high resolution, revealing novel targets for potential therapeutic candidate development.

Potential long-term effects of SARS-CoV-2 infection on the pulmonary vasculature: a global perspective.

The lungs are the primary target of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, with severe hypoxia being the cause of death in the most critical cases. Coronavirus disease 2019 (COVID-19) is extremely heterogeneous in terms of severity, clinical phenotype and, importantly, global distribution. Although the majority of affected patients recover from the acute infection, many continue to suffer from late sequelae affecting various organs, including the lungs. The role of the pulmonary vascular system during the acute and chronic stages of COVID-19 has not been adequately studied. A thorough understanding of the origins and dynamic behaviour of the SARS-CoV-2 virus and the potential causes of heterogeneity in COVID-19 is essential for anticipating and treating the disease, in both the acute and the chronic stages, including the development of chronic pulmonary hypertension. Both COVID-19 and chronic pulmonary hypertension have assumed global dimensions, with potential complex interactions. In this Review, we present an update on the origins and behaviour of the SARS-CoV-2 virus and discuss the potential causes of the heterogeneity of COVID-19. In addition, we summarize the pathobiology of COVID-19, with an emphasis on the role of the pulmonary vasculature, both in the acute stage and in terms of the potential for developing chronic pulmonary hypertension. We hope that the information presented in this Review will help in the development of strategies for the prevention and treatment of the continuing COVID-19 pandemic.

The integrated stress response in pulmonary disease.

The respiratory tract and its resident immune cells face daily exposure to stress, both from without and from within. Inhaled pathogens, including severe acute respiratory syndrome coronavirus 2, and toxins from pollution trigger a cellular defence system that reduces protein synthesis to minimise viral replication or the accumulation of misfolded proteins. Simultaneously, a gene expression programme enhances antioxidant and protein folding machineries in the lung. Four kinases (PERK, PKR, GCN2 and HRI) sense a diverse range of stresses to trigger this "integrated stress response". Here we review recent advances identifying the integrated stress response as a critical pathway in the pathogenesis of pulmonary diseases, including pneumonias, thoracic malignancy, pulmonary fibrosis and pulmonary hypertension. Understanding the integrated stress response provides novel targets for the development of therapies.

miR-1-5p targets TGF-ßR1 and is suppressed in the hypertrophying hearts of rats with pulmonary arterial hypertension.

The microRNA miR-1 is an important regulator of muscle phenotype including cardiac muscle. Down-regulation of miR-1 has been shown to occur in left ventricular hypertrophy but its contribution to right ventricular hypertrophy in pulmonary arterial hypertension are not known. Previous studies have suggested that miR-1 may suppress transforming growth factor-beta (TGF-ß) signalling, an important pro-hypertrophic pathway but only indirect mechanisms of regulation have been identified. We identified the TGF-ß type 1 receptor (TGF-ßR1) as a putative miR-1 target. We therefore hypothesized that miR-1 and TGF-ßR1 expression would be inversely correlated in hypertrophying right ventricle of rats with pulmonary arterial hypertension and that miR-1 would inhibit TGF-ß signalling by targeting TGF-ßR1 expression. Quantification of miR-1 and TGF-ßR1 in rats treated with monocrotaline to induce pulmonary arterial hypertension showed appropriate changes in miR-1 and TGF-ßR1 expression in the hypertrophying right ventricle. A miR-1-mimic reduced enhanced green fluorescent protein expression from a reporter vector containing the TGF-ßR1 3'- untranslated region and knocked down endogenous TGF-ßR1. Lastly, miR-1 reduced TGF-ß activation of a (mothers against decapentaplegic homolog) SMAD2/3-dependent reporter. Taken together, these data suggest that miR-1 targets TGF-ßR1 and reduces TGF-ß signalling, so a reduction in miR-1 expression may increase TGF-ß signalling and contribute to cardiac hypertrophy.

miR-322-5p targets IGF-1 and is suppressed in the heart of rats with pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is characterised by remodelling of the pulmonary vasculature leading to right ventricular hypertrophy. Here, we show that miR-322-5p (the rodent orthologue of miR-424-5p) expression is decreased in the right ventricle of monocrotaline-treated rats, a model of PAH, whereas a putative target insulin-like growth factor 1 (IGF-1) is increased. IGF-1 mRNA was enriched 16-fold in RNA immunoprecipitated with Ago2, indicating binding to miR-322-5p. In cell transfection experiments, miR-322-5p suppressed the activity of a luciferase reporter containing a section of the IGF-1 3' untranslated region (UTR) as well as IGF-1 mRNA and protein levels. Taken together, these data suggest that miR-322 targets IGF-1, a process downregulated in PAH-related RV hypertrophy.

Inhibition of hyaluronan synthesis attenuates pulmonary hypertension associated with lung fibrosis.

BACKGROUND AND PURPOSE: Group III pulmonary hypertension (PH) is a highly lethal and widespread lung disorder that is a common complication in idiopathic pulmonary fibrosis (IPF) where it is considered to be the single most significant predictor of mortality. While increased levels of hyaluronan have been observed in IPF patients, hyaluronan-mediated vascular remodelling and the hyaluronan-mediated mechanisms promoting PH associated with IPF are not fully understood. EXPERIMENTAL APPROACH: Explanted lung tissue from patients with IPF with and without a diagnosis of PH was used to identify increased levels of hyaluronan. In addition, an experimental model of lung fibrosis and PH was used to test the capacity of 4-methylumbeliferone (4MU), a hyaluronan synthase inhibitor to attenuate PH. Human pulmonary artery smooth muscle cells (PASMC) were used to identify the hyaluronan-specific mechanisms that lead to the development of PH associated with lung fibrosis. KEY RESULTS: In patients with IPF and PH, increased levels of hyaluronan and expression of hyaluronan synthase genes are present. Interestingly, we also report increased levels of hyaluronidases in patients with IPF and IPF with PH. Remarkably, our data also show that 4MU is able to inhibit PH in our model either prophylactically or therapeutically, without affecting fibrosis. Studies to determine the hyaluronan-specific mechanisms revealed that hyaluronan fragments result in increased PASMC stiffness and proliferation but reduced cell motility in a RhoA-dependent manner. CONCLUSIONS AND IMPLICATIONS: Taken together, our results show evidence of a unique mechanism contributing to PH in the context of lung fibrosis.