Hypoxia signaling plays a major role in non-malignant and malignant hyperproliferative diseases. Pulmonary hypertension (PH), a hypoxia-driven vascular disease, is characterized by a glycolytic ...switch similar to the Warburg effect in cancer. Ras association domain family 1A (RASSF1A) is a scaffold protein that acts as a tumour suppressor. Here we show that hypoxia promotes stabilization of RASSF1A through NOX-1- and protein kinase C- dependent phosphorylation. In parallel, hypoxia inducible factor-1 α (HIF-1α) activates RASSF1A transcription via HIF-binding sites in the RASSF1A promoter region. Vice versa, RASSF1A binds to HIF-1α, blocks its prolyl-hydroxylation and proteasomal degradation, and thus enhances the activation of the glycolytic switch. We find that this mechanism operates in experimental hypoxia-induced PH, which is blocked in RASSF1A knockout mice, in human primary PH vascular cells, and in a subset of human lung cancer cells. We conclude that RASSF1A-HIF-1α forms a feedforward loop driving hypoxia signaling in PH and cancer.
BACKGROUND:The angiogenic function of endothelial cells is regulated by numerous mechanisms, but the impact of long noncoding RNAs (lncRNAs) has hardly been studied. We set out to identify novel and ...functionally important endothelial lncRNAs.
METHODS:Epigenetically controlled lncRNAs in human umbilical vein endothelial cells were searched by exon-array analysis after knockdown of the histone demethylase JARID1B. Molecular mechanisms were investigated by RNA pulldown and immunoprecipitation, mass spectrometry, microarray, several knockdown approaches, CRISPR-Cas9, assay for transposase-accessible chromatin sequencing, and chromatin immunoprecipitation in human umbilical vein endothelial cells. Patient samples from lung and tumors were studied for MANTIS expression.
RESULTS:A search for epigenetically controlled endothelial lncRNAs yielded lncRNA n342419, here termed MANTIS, as the most strongly regulated lncRNA. Controlled by the histone demethylase JARID1B, MANTIS was downregulated in patients with idiopathic pulmonary arterial hypertension and in rats treated with monocrotaline, whereas it was upregulated in carotid arteries of Macaca fascicularis subjected to atherosclerosis regression diet, and in endothelial cells isolated from human glioblastoma patients. CRISPR/Cas9-mediated deletion or silencing of MANTIS with small interfering RNAs or GapmeRs inhibited angiogenic sprouting and alignment of endothelial cells in response to shear stress. Mechanistically, the nuclear-localized MANTIS lncRNA interacted with BRG1, the catalytic subunit of the switch/sucrose nonfermentable chromatin-remodeling complex. This interaction was required for nucleosome remodeling by keeping the ATPase function of BRG1 active. Thereby, the transcription of key endothelial genes such as SOX18, SMAD6, and COUP-TFII was regulated by ensuring efficient RNA polymerase II machinery binding.
CONCLUSION:MANTIS is a differentially regulated novel lncRNA facilitating endothelial angiogenic function.
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.
Pulmonary hypertension (PH), a progressive cardiopulmonary disease, is characterized by pathological remodeling of the pulmonary vasculature and increase in pulmonary vascular pressure resulting to ...its failure. Although numerous therapeutic options are now available to improve the quality of life for these patients, their life expectancy is still short. The underlying molecular mechanisms involved in the induction and progression of the disease phenotype are yet to be fully understood. Platelet-derived growth factor (PDGF) is a potent mitogen for vascular smooth muscle cells. Here, Savai et al cite the study by Deng and colleagues that describes the role of a novel PDGF-regulated lncRNA, lncPTSR, in controlling intracellular calcium levels and thereby contributing to the remodeling of vascular in PH.
Long noncoding RNAs (lncRNAs) are emerging as important regulators of diverse biological functions. Their role in pulmonary arterial hypertension (PAH) remains to be explored.
To elucidate the role ...of TYKRIL (tyrosine kinase receptor-inducing lncRNA) as a regulator of p53/ PDGFRβ (platelet-derived growth factor receptor β) signaling pathway and to investigate its role in PAH.
Pericytes and pulmonary arterial smooth muscle cells exposed to hypoxia and derived from patients with idiopathic PAH were analyzed with RNA sequencing. TYKRIL knockdown was performed in above-mentioned human primary cells and in precision-cut lung slices derived from patients with PAH.
Using RNA sequencing data, TYKRIL was identified to be consistently upregulated in pericytes and pulmonary arterial smooth muscles cells exposed to hypoxia and derived from patients with idiopathic PAH. TYKRIL knockdown reversed the proproliferative (
= 3) and antiapoptotic (
= 3) phenotype induced under hypoxic and idiopathic PAH conditions. Owing to the poor species conservation of TYKRIL,
studies were performed in precision-cut lung slices from patients with PAH. Knockdown of TYKRIL in precision-cut lung slices decreased the vascular remodeling (
= 5). The number of proliferating cell nuclear antigen-positive cells in the vessels was decreased and the number of terminal deoxynucleotide transferase-mediated dUTP nick end label-positive cells in the vessels was increased in the LNA (locked nucleic acid)-treated group compared with control. Expression of PDGFRβ, a key player in PAH, was found to strongly correlate with TYKRIL expression in the patient samples (
= 12), and TYKRIL knockdown decreased PDGFRβ expression (
= 3). From the transcription factor-screening array, it was observed that TYKRIL knockdown increased the p53 activity, a known repressor of PDGFRβ. RNA immunoprecipitation using various p53 mutants demonstrated that TYKRIL binds to the N-terminal of p53 (an important region for p300 interaction with p53). The proximity ligation assay revealed that TYKRIL interferes with the p53-p300 interaction (
= 3) and regulates p53 nuclear translocation.
TYKRIL plays an important role in PAH by regulating the p53/PDGFRβ axis.
The precise origin of newly formed ACTA2+ (alpha smooth muscle actin-positive) cells appearing in nonmuscularized vessels in the context of pulmonary hypertension is still debatable although it is ...believed that they predominantly derive from preexisting vascular smooth muscle cells (VSMCs).
mice were used to lineage trace GLI1+ (glioma-associated oncogene homolog 1-positive) cells in the context of pulmonary hypertension using 2 independent models of vascular remodeling and reverse remodeling: hypoxia and cigarette smoke exposure. Hemodynamic measurements, right ventricular hypertrophy assessment, flow cytometry, and histological analysis of thick lung sections followed by state-of-the-art 3-dimensional reconstruction and quantification using Imaris software were used to investigate the contribution of GLI1+ cells to neomuscularization of the pulmonary vasculature.
The data show that GLI1+ cells are abundant around distal, nonmuscularized vessels during steady state, and this lineage contributes to around 50% of newly formed ACTA2+ cells around these normally nonmuscularized vessels. During reverse remodeling, cells derived from the GLI1+ lineage are largely cleared in parallel to the reversal of muscularization. Partial ablation of GLI1+ cells greatly prevented vascular remodeling in response to hypoxia and attenuated the increase in right ventricular systolic pressure and right heart hypertrophy. Single-cell RNA sequencing on sorted lineage-labeled GLI1+ cells revealed an
fraction of cells with pathways in cancer and MAPK (mitogen-activated protein kinase) signaling as potential players in reprogramming these cells during vascular remodeling. Analysis of human lung-derived material suggests that GLI1 signaling is overactivated in both group 1 and group 3 pulmonary hypertension and can promote proliferation and myogenic differentiation.
Our data highlight GLI1+ cells as an alternative cellular source of VSMCs in pulmonary hypertension and suggest that these cells and the associated signaling pathways represent an important therapeutic target for further studies.
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 HIF1α-AS1 was retrieved as a top hit. Endogenous HIF1α-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 HIF1α-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 HIF1α-AS1, DNA:DNA:RNA triplex formation is a functionally important mechanism of trans-acting gene expression control.
IntroductionDespite substantial advancements in the treatment of pulmonary arterial hypertension (PAH), obstacles still remain in achieving the optimal outcomes. Various treatments have been ...developed to target signaling pathways mostly leading to increased intracellular cAMP. Existence of intracellular cAMP microenvironment and various cAMP regulators and effectors as signalosome, adds a different level of complexity to the cAMP signaling.MethodsIn-vitro studies involved mainly donor and IPAH human pulmonary artery smooth muscle cells (PASMCs). To study the complex we used co-immunoprecipitation, immunofluorescence staining and proximity ligation assay. Functional studies include performing proliferation and apoptosis assay after targeting ADORA1/PDE10A using siRNAs and inhibitors. cAMP levels were measured using ELISA kits. In-vivo studies involved treating the SU+Hypoxia and MCT PAH rat models with dual inhibitor followed by cardiac MRI, right heart catheterization and lung morphometric analysisResultsFrom the expression studies, it was observed that ADORA1 was highly expressed under the disease condition. Screening for cAMP inhibiting PDEs colocalised with ADORA1, it was observed PDE10A was in close proximity to ADORA1 compared to other cAMP targeting PDEs. PDE10A was also upregulated under the disease condition. From the functional studies it was observed that genetic and pharmacological inhibition of ADORA1 and PDE10A induced pro-apoptotic and anti-proliferative effect in human PASMCs via increased cAMP levels. Impressively, in-vivo studies, the dual inhibitor treated SU+hypoxia and MCT-PAH rats have shown better survival rate compared to the placebo. The hemodynamics and MRI data have shown that dual inhibitor treated rats were having lower pulmonary vascular resistance, reduced right ventricular hypertrophy and increased cardiac functioning. The morphometric analysis revealed that the rats treated with dual inhibitor had reduced medial wall thickness and muscularization compared to the placebo group.ConclusionThese results show that targeting ADORA1/PDE10A signalosome regulated cAMP microenvironment is a promising step towards having a better treatment in the field of pulmonary hypertension.
BackgroundIn recent years, understanding the role of epigenetic regulators in disease pathogenesis has gained interest. The role of long non coding RNA (LncRNA), which functions as epigenetic ...regulators are still to be explored in the field of pulmonary hypertension (PH). Pathological remodeling of pulmonary vessels and elevated pulmonary pressures are the major characteristics of PH. The pro-proliferative and anti-apoptotic phenotype of various resident and non-resident cells contribute to the vessel remodeling and molecular mechanism driving this phenotype is not well understood.ResultsUsing the RNAseq data, LncRNA TYKRIL (Tyrosine kinase receptor inducing LncRNA) 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 (60%± 2 reduction vs Ctrl, P<0.001, n=3) and anti-apoptotic (52%±3 increase vs Ctrl, P<0.001, 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 (40%±3 reduction vs Ctrl, P<0.001, n=3) and number of PCNA positive cells in the vessels (47%±3 reduction vs Ctrl, P<0.01, n=3). Expression of PDGFRβ, a key player in PH, was found to strongly correlate with TYKRIL expression in the patient samples (r=0.77, n=12) and TYKRIL knockdown decreased PDGFRβ expression (1.4±0.5 FC vs Ctrl, P<0.05, n=3). From the transcription factor-screening array, it was observed that TYKRIL knock down increased the p53 activity, a known repressor of PDGFRβ. Using RNA immunoprecipitation (RIP), it was found that TYKRIL interacts with p53 (TYKRIL enrichment FC 2.7±0.3 vs Ctrl, P<0.001, n=3). RIP using various p53 mutants demonstrated that TYKRIL binds to the N-terminal of p53 (an important region for p300 interaction with p53). The proximity ligation assay reveailed that TYKRIL interferes with the p53-p300 interaction (3.5±0.5 FC vs Ctrl, P<0.001, n=3) and regulates p53 nuclear translocation.ConclusionTYKRIL plays an important role in pulmonary hypertension by regulating the p53/PDGFRβ axis.
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