Jes-Niels Boeckel

Identification and Characterization of Hypoxia-Regulated Endothelial Circular RNA

RATIONALE Circular RNAs (circRNAs) are noncoding RNAs generated by back splicing. Back splicing has been considered a rare event, but recent studies suggest that circRNAs are widely expressed. However, the expression, regulation, and function of circRNAs in vascular cells is still unknown. OBJECTIVE Here, we characterize the expression, regulation, and function of circRNAs in endothelial cells. METHODS AND RESULTS Endothelial circRNAs were identified by computational analysis of ribo-minus RNA generated from human umbilical venous endothelial cells cultured under normoxic or hypoxic conditions. Selected circRNAs were biochemically characterized, and we found that the majority of them lacks polyadenylation, is resistant to RNase R digestion and localized to the cytoplasm. We further validated the hypoxia-induced circRNAs cZNF292, cAFF1, and cDENND4C, as well as the downregulated cTHSD1 by reverse transcription polymerase chain reaction in cultured endothelial cells. Cloning of cZNF292 validated the predicted back splicing of exon 4 to a new alternative exon 1A. Silencing of cZNF292 inhibited cZNF292 expression and reduced tube formation and spheroid sprouting of endothelial cells in vitro. The expression of pre-mRNA or mRNA of the host gene was not affected by silencing of cZNF292. No validated microRNA-binding sites for cZNF292 were detected in Argonaute high-throughput sequencing of RNA isolated by cross-linking and immunoprecipitation data sets, suggesting that cZNF292 does not act as a microRNA sponge. CONCLUSIONS We show that the majority of the selected endothelial circRNAs fulfill all criteria of bona fide circRNAs. The circRNA cZNF292 exhibits proangiogenic activities in vitro. These data suggest that endothelial circRNAs are regulated by hypoxia and have biological functions.

Heparin Selectively Affects the Quantification of MicroRNAs in Human Blood Samples

To the Editor: MicroRNAs (miRNAs)1 are noncoding RNA molecules that either inhibit translational processing or mediate degradation of target mRNAs in various physiological and pathophysiological processes (1). miRNAs have been detected in several body fluids, such as blood plasma, and have emerged as potentially suitable biomarkers for various diseases, including cancer and myocardial infarction (2). Heparin is commonly used as an anticoagulant in cardiovascular diagnostics and interventions. Previous studies have demonstrated that heparin can inhibit RNA quantification in vitro by interfering with the DNA polymerase used in the quantitative PCR (qPCR) reaction (3). It is unclear, however, whether the biologically active concentrations of heparin achieved after treatment of patients influences the quantification of miRNAs in blood samples. We therefore assessed the effect of systemic application of heparin on the measurement of endogenous circulating miRNAs in plasma samples. Blood samples from 11 patients were obtained before the cardiac catheterization procedure and at 10 and 60 min after heparin was administered (bolus, 5000 IU heparin; maintenance dose, 2500–5000 IU). We isolated RNA from EDTA-treated plasma with a TRIzol-based miRNA-isolation protocol (miRNeasy; Qiagen). We used a hydrolysis probe–based quantitative PCR method to measure …

Adenosine-to-inosine RNA editing controls cathepsin S expression in atherosclerosis by enabling HuR-mediated post-transcriptional regulation

Adenosine-to-inosine (A-to-I) RNA editing, which is catalyzed by a family of adenosine deaminase acting on RNA (ADAR) enzymes, is important in the epitranscriptomic regulation of RNA metabolism. However, the role of A-to-I RNA editing in vascular disease is unknown. Here we show that cathepsin S mRNA (CTSS), which encodes a cysteine protease associated with angiogenesis and atherosclerosis, is highly edited in human endothelial cells. The 3′ untranslated region (3′ UTR) of the CTSS transcript contains two inverted repeats, the AluJo and AluSx+ regions, which form a long stem–loop structure that is recognized by ADAR1 as a substrate for editing. RNA editing enables the recruitment of the stabilizing RNA-binding protein human antigen R (HuR; encoded by ELAVL1) to the 3′ UTR of the CTSS transcript, thereby controlling CTSS mRNA stability and expression. In endothelial cells, ADAR1 overexpression or treatment of cells with hypoxia or with the inflammatory cytokines interferon-γ and tumor-necrosis-factor-α induces CTSS RNA editing and consequently increases cathepsin S expression. ADAR1 levels and the extent of CTSS RNA editing are associated with changes in cathepsin S levels in patients with atherosclerotic vascular diseases, including subclinical atherosclerosis, coronary artery disease, aortic aneurysms and advanced carotid atherosclerotic disease. These results reveal a previously unrecognized role of RNA editing in gene expression in human atherosclerotic vascular diseases.

Hypoxia-induced alternative splicing in endothelial cells.

Background Adaptation to low oxygen by changing gene expression is vitally important for cell survival and tissue development. The sprouting of new blood vessels, initiated from endothelial cells, restores the oxygen supply of ischemic tissues. In contrast to the transcriptional response induced by hypoxia, which is mainly mediated by members of the HIF family, there are only few studies investigating alternative splicing events. Therefore, we performed an exon array for the genome-wide analysis of hypoxia-related changes of alternative splicing in endothelial cells. Methodology/Principal findings Human umbilical vein endothelial cells (HUVECs) were incubated under hypoxic conditions (1% O2) for 48 h. Genome-wide transcript and exon expression levels were assessed using the Affymetrix GeneChip Human Exon 1.0 ST Array. We found altered expression of 294 genes after hypoxia treatment. Upregulated genes are highly enriched in glucose metabolism and angiogenesis related processes, whereas downregulated genes are mainly connected to cell cycle and DNA repair. Thus, gene expression patterns recapitulate known adaptations to low oxygen supply. Alternative splicing events, until now not related to hypoxia, are shown for nine genes: six which are implicated in angiogenesis-mediated cytoskeleton remodeling (cask, itsn1, larp6, sptan1, tpm1 and robo1); one, which is involved in the synthesis of membrane-anchors (pign) and two universal regulators of gene expression (cugbp1 and max). Conclusions/Significance For the first time, this study investigates changes in splicing in the physiological response to hypoxia on a genome-wide scale. Nine alternative splicing events, until now not related to hypoxia, are reported, considerably expanding the information on splicing changes due to low oxygen supply. Therefore, this study provides further knowledge on hypoxia induced gene expression changes and presents new starting points to study the hypoxia adaptation of endothelial cells.

MicroRNAs in Platelet Biogenesis and Function: Implications in Vascular Homeostasis and Inflammation

Platelets are involved in vascular homeostasis and inflammation through interaction with circulating blood cells and vascular wall. MiRNAs are small, conserved and non-coding RNA molecules, which interact directly with specific mRNAs regions regulating gene expression. The purpose of this review is to gather all known platelet miRNAs and summarize their role in platelet biogenesis and function. Increasing evidence supports the role of miR-34a and miR-150 in megakaryocytopoiesis and platelet production. Although 284 miRNAs are described to be present in platelets, their role is mostly unknown. The most abundant miRNA in platelets is miR-223 followed by miR-126. The miR-96, miR-200b, miR- 495, miR-107 and miR-223 are critically involved in platelet reactivity, aggregation, secretion and adhesion. The presence of miRNAs known to regulate angiogenesis in platelets is also discussed. Furthermore, platelet-derived microvesicles and microparticles contain several miRNAs, which may facilitate the communication between platelets with other vascular cells, a mechanism that may play an important role in vascular homeostasis and inflammation. Further studies are needed to elucidate the exact roles of platelet miRNAs in platelet function and vascular biology.

Transcoronary gradients of vascular miRNAs and coronary atherosclerotic plaque characteristics

AIMS Circulating microRNAs (miRs) may reflect pathophysiologically relevant processes in the atherosclerotically diseased coronary arterial wall. Given the unmet medical need to identify patients with an unstable plaque phenotype, we determined the relation of circulating atherosclerosis-regulatory miRs with plaque phenotypes. METHODS AND RESULTS We assessed coronary atherosclerotic plaque burden and phenotype by optical coherence tomography in 52 patients and measured the levels of circulating miRs across the transcoronary gradient. The overall plaque load was significantly correlated with transcoronary concentration gradients of miR-126-3p (P = 0.04), miR-145-5p (P = 0.01), miR-155-5p (P < 0.01), and miR-29b-3p (P = 0.02), but not with other miRs such as miR-92a-3p. In patients with a high extent of vulnerable plaques as assessed by the presence of thin-cap fibroatheromas (TCFAs), significantly higher transcoronary gradients were observed, particularly for miR-126-3p, miR-126-5p, and miR-145-5p (all P < 0.02). Transcoronary gradients of miR-126-3p (P < 0.01), miR-126-5p (P < 0.01), miR-145-5p (P = 0.01), miR-29b-3p (P = 0.03), and miR-155-5p (P = 0.02) demonstrated a significant discriminatory power to predict the presence of TCFAs (AUC > 0.7 for all). Moreover, aortic and venous coronary sinus levels of miR-29b-3p were inversely correlated with plaque fibrosis, a finding that is consistent with the anti-fibrotic activity of miR-29b-3p. CONCLUSION The overall plaque burden and plaque phenotypes are associated with changes in the kinetics of miR-concentrations across the transcoronary passage. Transcoronary gradients of the anti-atherosclerotic miR-126-3p and miR-145-5p correlated with the extent of TCFAs, suggesting that instable plaques may affect the local uptake or degradation of these miRs.

Coronary Atherosclerotic Plaque Characteristics and Cardiovascular Risk Factors ― Insights From an Optical Coherence Tomography Study ―

BACKGROUND The association between cardiovascular risk factors (CVRF) and the risk of coronary events is widely acknowledged. Whether individual risk factors may be associated with distinct plaque characteristics is currently unclear. We investigated the relationship between CVRF and coronary plaque burden and phenotype.Methods and Results:We assessed coronary atherosclerotic plaque characteristics by optical coherence tomography in 67 patients with stable coronary artery disease undergoing coronary angiography. The plaque burden and the distinct plaque phenotypes were compared with regard to different CVRF. Overall plaque burden was significantly greater in patients with diabetes mellitus (P=0.010), prediabetes (P=0.035) and obesity (P=0.024), and correlated with the number of CVRF (R=0.358, P=0.003). Patients with diabetes had a greater extent of fibroatheroma (P=0.015), calcific fibroatheroma (P=0.031), thin-cap fibroatheroma (TCFA-P=0.011) and plaque erosion (P=0.002). Obese patients showed a greater extent of fibroatheroma (P=0.007), TCFA (P=0.015) and macrophage load (P=0.043). The number of CVRF correlated with fibroatheroma (R=0.425, P<0.001), calcific fibroatheroma (R=0.321, P=0.008), TCFA (R=0.347, P=0.004), macrophage load (R=0.314, P=0.010) and erosion (R=0.271, P=0.029). In the multivariate analysis, altered glycemic status and obesity were the only independent predictors of TCFA (P=0.026 and P=0.046, respectively), whereas altered glycemic status was the only independent predictor of plaque erosion (P=0.001). CONCLUSIONS Patients with diabetes, prediabetes and obesity show more extensive coronary atherosclerosis and more vulnerable plaque phenotypes.

Identification and Functional Characterization of Hypoxia-Induced Endoplasmic Reticulum Stress Regulating lncRNA (HypERlnc) in PericytesNovelty and Significance

Rationale: Pericytes are essential for vessel maturation and endothelial barrier function. Long noncoding RNAs regulate many cellular functions, but their role in pericyte biology remains unexplored. Objective: Here, we investigate the effect of hypoxia-induced endoplasmic reticulum stress regulating long noncoding RNAs (HypERlnc, also known as ENSG00000262454) on pericyte function in vitro and its regulation in human heart failure and idiopathic pulmonary arterial hypertension. Methods and Results: RNA sequencing in human primary pericytes identified hypoxia-regulated long noncoding RNAs, including HypERlnc. Silencing of HypERlnc decreased cell viability and proliferation and resulted in pericyte dedifferentiation, which went along with increased endothelial permeability in cocultures consisting of human primary pericyte and human coronary microvascular endothelial cells. Consistently, Cas9-based transcriptional activation of HypERlnc was associated with increased expression of pericyte marker genes. Moreover, HypERlnc knockdown reduced endothelial-pericyte recruitment in Matrigel assays (P<0.05). Mechanistically, transcription factor reporter arrays demonstrated that endoplasmic reticulum stress-related transcription factors were prominently activated by HypERlnc knockdown, which was confirmed via immunoblotting for the endoplasmic reticulum stress markers IRE1&agr; (P<0.001), ATF6 (P<0.01), and soluble BiP (P<0.001). Kyoto encyclopedia of genes and gene ontology pathway analyses of RNA sequencing experiments after HypERlnc knockdown indicate a role in cardiovascular disease states. Indeed, HypERlnc expression was significantly reduced in human cardiac tissue from patients with heart failure (P<0.05; n=19) compared with controls. In addition, HypERlnc expression significantly correlated with pericyte markers in human lungs derived from patients diagnosed with idiopathic pulmonary arterial hypertension and from donor lungs (n=14). Conclusions: Here, we show that HypERlnc regulates human pericyte function and the endoplasmic reticulum stress response. In addition, RNA sequencing analyses in conjunction with reduced expression of HypERlnc in heart failure and correlation with pericyte markers in idiopathic pulmonary arterial hypertension indicate a role of HypERlnc in human cardiopulmonary disease.

Analyzing the Release of Copeptin from the Heart in Acute Myocardial Infarction Using a Transcoronary Gradient Model

Copeptin is the C-terminal end of pre-provasopressin released equimolar to vasopressin into circulation and recently discussed as promising cardiovascular biomarker amendatory to established markers such as troponins. Vasopressin is a cytokine synthesized in the hypothalamus. A direct release of copeptin from the heart into the circulation is implied by data from a rat model showing a cardiac origin in hearts put under cardiovascular wall stress. Therefore, evaluation of a potential release of copeptin from the human heart in acute myocardial infarction (AMI) has been done.

Estimation of Values below the Limit of Detection of a Contemporary Sensitive Troponin I Assay Improves Diagnosis of Acute Myocardial Infarction

BACKGROUND The limit of detection (LoD) is the minimal amount of a substance that can be consistently detected. In the diagnosis of acute myocardial infarction (AMI) many patients present with troponin concentrations below the LoD of contemporary sensitive cardiac troponin I (cs-cTnI) assays. These censored values below the LoD influence the diagnostic performance of these assays compared to highly sensitive cTnI (hs-cTnI) assays. Therefore we assessed the impact of a new approach for interpolation of the left-censored data of a cs-cTnI assay in the evaluation of patients with suspected AMI. METHODS Our posthoc analysis used a real world cohort of 1818 patients with suspected MI. Data on cs-cTnI was available in 1786 patients. As a comparator the hs-cTnI version of the assay was used. To reconstruct quantities below the LoD of the cs-cTnI assay, a gamma regression approach incorporating the GRACE (Global Registry of Acute Coronary Events) score variables was used. RESULTS Censoring of cs-cTnI data below the LoD yielded weaker diagnostic information [area under the curve (AUC), 0.781; 95% CI, 0.731-0.831] regarding AMI compared to the hs-cTnI assay (AUC, 0.949; CI, 0.936-0.961). Use of our model to estimate cs-cTnI values below the LoD showed an AUC improvement to 0.921 (CI, 0.902-0.940). The cs-cTnI LoD concentration had a negative predictive value (NPV) of 0.950. An estimated concentration that was to be undercut by 25% of patients presenting with suspected AMI was associated with an improvement of the NPV to 0.979. CONCLUSIONS Estimation of values below the LoD of a cs-cTnI assay with this new approach improves the diagnostic performance in evaluation of patients with suspected AMI.