Non-invasive approach for evaluation of CTEPH using extracellular vesicle-associated small non-coding RNA
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ABSTRACT: The aim of this study was to determine whether extracellular vesicle (EV)-associated small non-coding RNAs (sncRNAs) have potential as biomarkers for chronic thromboembolic pulmonary hypertension (CTEPH). EVs were isolated using different methods from serum of 23 CTEPH patients and 23 controls. EV-associated RNAs were analysed by next-generation sequencing using the TrueQuant method for molecular barcoding, and differentially expressed sncRNAs were validated by qRT-PCR. We identified 18 miRNAs and 21 piRNAs or piRNA clusters that were differentially expressed in CTEPH patients compared with the control group. Bioinformatic analysis predicted a contribution of these piRNAs to the progression of cardiac and vascular remodelling. Furthermore, the expression levels of DQ593039 correlated with clinically meaningful parameters such as mean pulmonary arterial pressure, pulmonary vascular resistance, right ventricular systolic pressure, and levels of N-terminal pro-brain natriuretic peptide. In summary, EV-associated piRNA DQ593039 shows promise as biomarker and may be a potential therapeutic target for CTEPH.
Project description:The hallmark of chronic thromboembolic pulmonary hypertension (CTEPH) is fibrotic transformation of pulmonary arterial thrombus, leading to mechanical obstruction of pulmonary arteries. We report the transcriptome profile of fibroblasts isolated from pairs of CTEPH thrombus and pulmonary artery adventitia of the same patient. Fibroblasts isolated from CTEPH thrombus demonstrated upregulation of genes associated with the TGF-β pathway indicating that TGF-β upregulation is associated with the intravascular remodeling process.
Project description:Chronic thromboembolic pulmonary hypertension (CTEPH) is a group 4 pulmonary hypertension (PH) characterized by non-resolving thromboembolism in the central pulmonary artery and vascular occlusion in the proximal and distal pulmonary artery. Medical therapy is chosen for patients who are ineligible for pulmonary endarterectomy or balloon pulmonary angioplasty or who have symptomatic residual PH after surgery or intervention. Selexipag, an oral prostacyclin receptor agonist and potent vasodilator, was approved for CTEPH in Japan in 2021. To evaluate the pharmacological effect of selexipag on vascular occlusion in CTEPH, we examined how its active metabolite MRE-269 affects platelet-derived growth factor-stimulated pulmonary arterial smooth muscle cells (PASMCs) from CTEPH patients. MRE-269 showed a more potent anti-proliferative effect on PASMCs from CTEPH patients than on those from normal subjects. DNA-binding protein inhibitor genes ID1 and ID3 were found by RNA sequencing and real-time quantitative polymerase chain reaction to be expressed at lower levels in PASMCs from CTEPH patients than in those from normal subjects and were upregulated by MRE-269 treatment. ID1 and ID3 upregulation by MRE-269 was blocked by co-incubation with a prostacyclin receptor antagonist, and ID1 knockdown by small interfering RNA transfection attenuated the anti-proliferative effect of MRE-269. ID signaling may be involved in the anti-proliferative effect of MRE-269 on PASMCs. This is the first study to demonstrate the pharmacological effects on PASMCs from CTEPH patients of a drug approved for the treatment of CTEPH. Both the vasodilatory and the anti-proliferative effect of MRE-269 may contribute to the efficacy of selexipag in CTEPH.
Project description:PIWI-interacting RNAs (piRNAs) are thought to silence transposon and gene expression during development. However, the roles of piRNAs in somatic tissues are largely unknown. Here we report the identification of 555 piRNAs in human lung bronchial epithelial (HBE) and non-small cell lung cancer (NSCLC) cell lines, including 295 that don’t exist in databases termed as piRNA-Like sncRNAs or piRNA-Ls. Distinctive piRNA/piRNA-L expression patterns are observed between HBE and NSCLC cells. piRNA-L-163 (piR-L-163), the top down-regulated piRNA-L in NSCLC cells, binds directly to phosphorylated ERM proteins (p-ERM), which is dependent on the central part of UUNNUUUNNUU motif in piR-L-163 and the RRRKPDT element in ERM, and. The piR-L-163/p-ERM interaction is critical for p-ERM’s binding capability to filamentous actin (F-actin) and ERM-binding phosphoprotein 50 (EBP50). Thus, piRNA/piRNA-L may play a regulatory role through direct interaction with proteins in physiological and pathophysiological conditions.
Project description:Our current understanding of the pathobiology of chronic thromboembolic pulmonary hypertension (CTEPH) is primarily based on studies using specimens obtained through pulmonary endarterectomy (PEA). However, there is a significant gap in our knowledge regarding the characteristics and functions of circulating immune cells in CTEPH patients. To address this gap, our study aims to characterize the peripheral blood circulating immune cells in CTEPH patients, with a particular focus on monocytes. In this study, we performed single-cell RNA sequencing (scRNA-seq) on peripheral blood mononuclear cells (PBMCs) extracted from five CTEPH patients. This approach allowed us to identify various immune cell types present in the peripheral blood, including monocytes, T cells, and B cells. Notably, we observed an increased proportion of CD16+ monocytes in CTEPH patients. This monocyte subset exhibited distinct transcriptional profiles, indicating their involvement in critical biological processes such as cell adhesion, T cell activation, coagulation, and platelet activation. Our findings suggest that CD16+ monocytes may play a significant role in the pathogenesis of CTEPH, particularly in pulmonary arterial thrombosis and intimal remodeling. Furthermore, we observed that this subset of monocytes might be recruited into the pulmonary artery intima and differentiate into macrophages characterized by high IL-1β expression, potentially contributing to disease progression. This study provides new insights into the immune landscape of CTEPH and highlights the potential role of CD16+ monocytes in the disease's pathophysiology.
Project description:The present prospective study included a total number of 3 patients with a final diagnosis of chronic thromboembolic pulmonary hypertension (CTEPH), who were treated by pulmonary endarterectomy (PEA) at the Kerckhoff Heart and Thorax Center between 2016 and 2020. Biopsies of the myocardial interventricular septum from 3 patients were collected at baseline (BL) before PEA surgery (pre-septal-PEA) In this case, to account for technical and safety aspects, the specimens were taken from the myocardial interventricular septum. The aim of the Study was a comparative characterization of RNA-profiles of septum in CTEPH patients after PEA surgery (postPEA) compared to before PEA surgery (pre-septal-PEA).
Project description:The present prospective study included a total number of 9 patients with a final diagnosis of chronic thromboembolic pulmonary hypertension (CTEPH), who were treated by pulmonary endarterectomy (PEA) at the Kerckhoff Heart and Thorax Center between 2016 and 2020. Biopsies of theright ventricularfreewall from9patients were collected at baseline (BL) before PEA surgery (prePEA). Patients were grouped in severe and moderate risk groups according to 1 year mortality risk by applying a modified version of the European Society of Cardiology (ESC) guidelines risk stratification model according to their BL characteristics. The aim of the Study was a comparative characterization of RNA-profiles in CTEPH patients with disease status at baseline prior to PEA therapy
Project description:Hepatocellular carcinoma (HCC) is considered the result of a stepwise carcinogenic process, more often beginning with development of premalignant lesions within a cirrhotic liver, morphologically characterized by low- (LGDN) and high-grade (HGDN) dysplastic nodules. Piwi-interacting RNAs (piRNAs) represent a large family of small noncoding (snc) RNAs, 23-35 nucleotide-long, first identified in fruitfly germline cells by their ability to interact with PIWI proteins and thereby exert epigenetic and post-transcriptional regulation of gene expression. The PIWI-piRNAs pathway is active also in human somatic tissues, including stem and cancer cells, where piRNAs and piRNA-like molecules have been shown to influence key cellular processes. We applied small RNA sequencing to search for human liver piRNAs and to profile their expression patterns in cirrhotic nodules (CNs), LGDN, HGDN, early HCC (eHCC) and progressed HCC (pHCC), aiming at identifying possible relationships between these sncRNAs and liver carcinogenesis. Analyzing 54 nodules (14 CN, 8 LGDN, 6 HGDN, 6 eHCC and 20 pHCC) from 17 patients, we identified a 125 piRNA expression signature that clearly discriminates HCC from matched CNs, correlating also to clinicopathological characteristics of HCC. This result was confirmed by functional analysis of predicted piRNA target mRNAs. Interestingly, 24 piRNAs showed specific expression patterns in dysplastic nodules, respect to cirrhotic liver and/or pHCC. These results demonstrate that the piRNA pathway is active in human liver, where it is likely to represent a new player in the molecular events that characterize hepatocellular carcinogenesis, from early stages to pHCC. Furthermore, they suggest that piRNAs might represent new disease biomarkers, potentially useful for differential diagnosis of dysplastic and neoplastic liver lesions.
Project description:The present prospective study included a total number of 71 patients with a final diagnosis of chronic thromboembolic pulmonary hypertension (CTEPH), who were treated by pulmonary endarterectomy (PEA) at the Kerckhoff Heart and Thorax Center between 2016 and 2020. Biopsies of the free right ventricular wall from 71 patients were collected at baseline (BL) during PEA. In a subgroup of 24 patients, myocardial interventricular septum biopsie were obtained during right heart catheterization (RHC) 12 months after PEA (postPEA) In this case, to account for technical and safety aspects, the specimens were taken from the myocardial interventricular septum. Patients were grouped in severe, intermediat and moderate risk according to 1 year mortality risk by applying a modified version of the European Society of Cardiology (ESC) guidelines risk stratification model according to their BL characteristics. The aim of the Study was: a) A comparative characterization of RNA-profiles in CTEPH patients with disease status at baseline prior to PEA therapy; b): An assessment of dynamics of the RNA-profiles in CTEPH patients pre and post PEA.
Project description:Piwi-interacting RNAs (piRNAs) are a distinct group of small non-coding RNAs (sncRNAs) that silence transposable genetic elements to protect genome integrity. Due to their limited expression in gonads and sequence diversity, piRNAs remain the most mysterious class of small RNAs. Studies have shown piRNAs are present in somatic cells and dysregulated in gastric, breast and liver cancers. By deep sequencing 24 frozen benign kidney and clear cell renal cell carcinoma (ccRCC) specimens and using the publically available piRNA database, we found 26,991 piRNAs present in human kidney tissue. Among 920 piRNAs that at least had two copies in one specimen, 19 were differentially expressed in benign kidney and ccRCC tissues, and 46 were associated with metastasis. Among the metastasis-related piRNAs, we found 3 piRNAs, piR- 32051, piR-39894 and piR-43607, to be derived from the same piRNA cluster at chromosome 17. We confirmed the 3 selected piRNAs not to be miRNAs or miRNA-like sncRNAs. We further validated the aberrant expression of the 3 piRNAs in a 68-case formalin-fixed and paraffin-embedded (FFPE) ccRCC tissue cohort and showed the upregulation of the 3 piRNAs to be highly associated with ccRCC metastasis, late clinical stage and cancer-specific survival.
Project description:PIWI-clade Argonaute proteins silence transposon expression in animal gonads. Their target specificity is defined by bound ~23-30nt piRNAs that are processed from single-stranded precursor transcripts via two distinct pathways. Primary piRNAs are defined by the endo-nuclease Zucchini, while biogenesis of secondary piRNAs depends on piRNA-guided transcript cleavage and results in piRNA amplification. Here, we analyze the inter-dependencies between these piRNA biogenesis pathways in the developing Drosophila ovary. We show that secondary piRNA-guided target slicing is the predominant mechanism that specifies transcripts—including those from piRNA clusters—as primary piRNA precursors and that defines the spectrum of Piwi-bound piRNAs in germline cells. Post-transcriptional silencing in the cytoplasm therefore enforces nuclear, transcriptional target silencing, which ensures the tight suppression of transposons during oogenesis. As target slicing also defines the nuclear piRNA pool during mouse spermatogenesis, our findings uncover an unexpected conceptual similarity between the mouse and fly piRNA pathways.