Transcriptional profiling unveils molecular subgroups of adaptive and maladaptive right ventricular remodeling in pulmonary hypertension

Right ventricular (RV) function is critical to prognosis in all forms of pulmonary hypertension. Here we perform molecular phenotyping of RV remodeling by transcriptome analysis of RV tissue obtained from 40 individuals, and two animal models of RV dysfunction of both sexes. Our unsupervised clustering analysis identified ‘early’ and ‘late’ subgroups within compensated and decompensated states, characterized by the expression of distinct signaling pathways, while fatty acid metabolism and estrogen response appeared to underlie sex-specific differences in RV adaptation. The circulating levels of several extracellular matrix proteins deregulated in decompensated RV subgroups were assessed in two independent cohorts of individuals with pulmonary arterial hypertension, revealing that NID1, C1QTNF1 and CRTAC1 predicted the development of a maladaptive RV state, as defined by magnetic resonance imaging parameters, and were associated with worse clinical outcomes. Our study provides a resource for subphenotyping RV states, identifying state-specific biomarkers, and potential therapeutic targets for RV dysfunction

Epigenetic reactivation of transcriptional programs orchestrating fetal lung development in human pulmonary hypertension

Phenotypic alterations in resident vascular cells contribute to the vascular remodeling process in diseases such as pulmonary (arterial) hypertension [P(A)H]. How the molecular interplay between transcriptional coactivators, transcription factors (TFs), and chromatin state alterations facilitate the maintenance of persistently activated cellular phenotypes that consequently aggravate vascular remodeling processes in PAH remains poorly explored. RNA sequencing (RNA-seq) in pulmonary artery fibroblasts (FBs) from adult human PAH and control lungs revealed 2460 differentially transcribed genes. Chromatin immunoprecipitation sequencing (ChIP-seq) revealed extensive differential distribution of transcriptionally accessible chromatin signatures, with 4152 active enhancers altered in PAH-FBs. Integrative analysis of RNA-seq and ChIP-seq data revealed that the transcriptional signatures for lung morphogenesis were epigenetically derepressed in PAH-FBs, including coexpression of T-box TF 4 (TBX4), TBX5, and SRY-box TF 9 (SOX9), which are involved in the early stages of lung development. These TFs were expressed in mouse fetuses and then repressed postnatally but were maintained in persistent PH of the newborn and reexpressed in adult PAH. Silencing of TBX4, TBX5, SOX9, or E1A-associated protein P300 (EP300) by RNA interference or small-molecule compounds regressed PAH phenotypes and mesenchymal signatures in arterial FBs and smooth muscle cells. Pharmacological inhibition of the P300/CREB-binding protein complex reduced the remodeling of distal pulmonary vessels, improved hemodynamics, and reversed established PAH in three rodent models in vivo, as well as reduced vascular remodeling in precision-cut tissue slices from human PAH lungs ex vivo. Epigenetic reactivation of TFs associated with lung development therefore underlies PAH pathogenesis, offering therapeutic opportunities

Fibrocytes boost tumor-supportive phenotypic switches in the lung cancer niche via the endothelin system

Fibrocytes are bone marrow-derived monocytic cells implicated in wound healing. Here, we identify their role in lung cancer progression/ metastasis. Selective manipulation of fibrocytes in mouse lung tumor models documents the central role of fibrocytes in boosting niche features and enhancing metastasis. Importantly, lung cancer patients show increased number of circulating fibrocytes and marked fibrocyte accumulation in the cancer niche. Using double and triple co-culture systems with human lung cancer cells, fibrocytes, macrophages and endothelial cells, we substantiate the central features of cancer-supporting niche: enhanced cancer cell proliferation and migration, macrophage activation, augmented endothelial cell sprouting and fibrocyte maturation. Upregulation of endothelin and its receptors are noted, and dual endothelin receptor blockade suppresses all cancer-supportive phenotypic alterations via acting on fibrocyte interaction with the cancer niche. We thus provide evidence for a crucial role of fibrocytes in lung cancer progression and metastasis, suggesting targets for treatment strategies. Fibrocytes are monocyte-derived cells implicated in wound healing. Here, the authors utilise single cell RNA-seq, genetic ablation and multiplexed imaging to identify a fibrocyte population in lung cancer models, and use human lung cancer coculture systems to highlight their potential to modulate microenvironmental niche and sensitivity to endothelin blockade

HIF1 alpha-AS1 is a DNA:DNA:RNA triplex-forming lncRNA interacting with the HUSH complex

DNA:DNA:RNA triplexes that are formed through Hoogsteen base-pairing of the RNA in the major groove of the DNA duplex have been observed in vitro, but the extent to which these interactions occur in cells and how they impact cellular functions remains elusive. Using a combination of bioinformatic techniques, RNA/DNA pulldown and biophysical studies, we set out to identify functionally important DNA:DNA:RNA triplex-forming long non-coding RNAs (lncRNA) in human endothelial cells. The lncRNA HIF1 alpha-AS1 was retrieved as a top hit. Endogenous HIF1 alpha-AS1 reduces the expression of numerous genes, including EPH Receptor A2 and Adrenomedullin through DNA:DNA:RNA triplex formation by acting as an adapter for the repressive human silencing hub complex (HUSH). Moreover, the oxygen-sensitive HIF1 alpha-AS1 is down-regulated in pulmonary hypertension and loss-of-function approaches not only result in gene de-repression but also enhance angiogenic capacity. As exemplified here with HIF1 alpha-AS1, DNA:DNA:RNA triplex formation is a functionally important mechanism of trans-acting gene expression control. Using a composite bioinformatics approach, the DNA:DNA:RNA triplex-forming lncRNAs HIF1 alpha-AS1 was identified in human endothelial cells which recruits an epigenetic silencing complex to limit expression of triplex target genes

Long noncoding RNA TYKRIL plays a role in pulmonary hypertension via the p53-mediated regulation of PDGFRβ

RATIONALE: Long noncoding RNAs (lncRNAs) are emerging as important regulators of diverse biological functions. Their role in pulmonary arterial hypertension (PAH) remains to be explored. OBJECTIVES: To elucidate the role of tyrosine kinase receptor inducing lncRNA (TYKRIL) as a regulator of p53/platelet-derived growth factor receptor β (PDGFRβ) signaling pathway and to investigate its role in PAH. MEASUREMENTS AND MAIN RESULTS: Using RNAseq data, TYKRIL was identified to be consistently upregulated in pericytes and pulmonary arterial smooth muscles cells (PASMCs) exposed to hypoxia and derived from IPAH patients. TYKRIL knockdown reversed the pro-proliferative (n=3) and anti-apoptotic (n=3) phenotype induced under hypoxic and IPAH conditions. Due to the poor species conservation of TYKRIL, ex-vivo studies were carried out in precision cut lung slices (PCLS) from PH patients. Knockdown of TYKRIL in PCLS decreased the vascular remodeling (n=5). The number of PCNA positive cells in the vessels were decreased and number of TUNEL positive cells in the vessels were increased in LNA treated group compared to control. Expression of PDGFRβ, a key player in PH, was found to strongly correlate with TYKRIL expression in the patient samples (n=12) and TYKRIL knockdown decreased PDGFRβ expression (n=3). Importantly, TYKRIL knockdown increased the p53 activity, a known repressor of PDGFRβ by binding to the N-terminal of p53 and interfering with p53-p300 interaction that subsequently regulates p53 nuclear translocation. CONCLUSION: TYKRIL plays an important role in PAH by regulating the p53/PDGFRβ axis