In acute stroke, timely diagnosis is essential to prevent extensive neuronal damage and improve patient outcomes. However, differentiating between ischemic stroke, intracerebral hemorrhage, and stroke mimics remains challenging. Transfer RNA-derived fragments (tRFs) have emerged as potential biomarkers for distinguishing between these stroke subtypes. Here we used reverse transcription-quantitative PCR (RT-qPCR) to investigate the expression of specific tRFs that we previously identified in small RNA sequencing data as potential biomarkers. Out of 12 measured tRFs, only the fragments ArgTCG53-67 and TyrGTA1-19 showed a trend for differential expression between stroke subtypes, but with insufficient predictive value to be of use in clinical practice. Combining expression data of specific tRFs into a joint model did not improve the predictive ability. Technical and computational challenges may compromise the reliability of tRF expression data from RNA sequencing, possibly explaining our inability to validate the specific tRFs as potential stroke biomarkers. Moreover, the limitations of RT-qPCR challenge a reliable quantification of these fragments even further. Our findings highlight the need for improved quantitative methods for tRF analysis to fully exploit their potential as clinically meaningful biomarkers. Addressing these technical barriers could unlock the diagnostic potential of tRFs, facilitating faster, more accurate stroke subtype identification in an acute setting.

BACKGROUND AND PURPOSE:Intracranial atherosclerosis is one of the most common causes of ischaemic stroke. However, there is a substantial knowledge gap on the development of intracranial atherosclerosis. Intracranial arteries are characterized by an upregulation of tight junctions between endothelial cells, which control endothelial permeability. We investigated the role of N6-methyladenosine (m6A), a common RNA modification, on endothelial integrity, focusing on the pro-atherogenic microRNA miR-494-3p and tight junction proteins TJP1 and PECAM1. EXPERIMENTAL APPROACH:We assessed the m6A landscape, along with the expression of miR-494-3p, TJP1 and PECAM1 in postmortem human vertebral arteries (VA), internal carotid arteries (ICA), and middle cerebral arteries (MCA) with various stages of intimal thickening and plaque formation. The interactions between m6A-modified miR-494-3p mimics, TJP1 and PECAM1, were investigated in vitro using primary human (brain) endothelial cells. KEY RESULTS:Increased m6A expression was observed in the luminal lining of atherosclerosis-affected VAs, accompanied by reduced TJP1 and PECAM1, but not VE-cadherin, expression. Colocalization of m6A and miR-494-3p in the luminal lining of VA plaques was confirmed, indicating m6A methylation of miR-494-3p in intracranial atherosclerosis. Moreover, site-specific m6A-modification of miR-494-3p led to repression specifically of TJP1 protein expression at cell-cell junctions of brain microvascular endothelial cells, while unmodified miR-494-3p showed no effect. CONCLUSIONS AND IMPLICATIONS:This study highlights increasing m6A levels during intracranial atherogenesis. Increases in m6A-miR-494-3p contribute to the observed decreased TJP1 expression in endothelial cell-cell junctions. This is likely to have a negative effect on endothelial integrity and may thus accelerate intracranial atherosclerosis progression.

Purpose of Review Small non-coding RNAs regulate gene expression and are highly implicated in heart failure. Recently, an additional level of post-transcriptional regulation has been identified, referred to as the epitranscriptome, which encompasses the body of post-transcriptional modifications that are placed on RNA molecules. In this review, we summarize the current knowledge on the small non-coding RNA epitranscriptome in heart failure. Recent Findings With the rise of new methods to study RNA modifications, epitranscriptome research has begun to take flight. Over the past 3 years, the number of publications on the epitranscriptome in heart failure has significantly increased, and we expect many more highly relevant publications to come out over the next few years. Summary Currently, at least six modifications on small non-coding RNAs have been investigated in heart failure-relevant studies, namely N6-adenosine, N5-cytosine and N7-guanosine methylation, 2’- O -ribose-methylation, adenosine-to-inosine editing, and isomiRs. Their potential role in heart failure is discussed.

C/D box small nucleolar RNAs (snoRNAs) of the DLK1-DIO3 locus are associated with vascular remodeling and cardiovascular disease. None of these snoRNAs has any known targets yet except for one, AF357425/SNORD113-6. We previously showed that this snoRNA targets mRNAs of the integrin signaling pathway and affects arterial fibroblast function. Here, we aimed to identify whether AF357425/SNORD113-6 can also target small RNAs. We overexpressed or inhibited AF357425 in murine fibroblasts and performed small RNA sequencing. Expression of transfer (t)RNA fragments (tRFs) was predominantly regulated. Compared with overexpression, AF357425 knockdown led to an overall decrease in tRFs but with an enrichment in smaller tRFs (<30 nucleotides). We focused on tRNA leucine anti-codon TAA (tRNALeu(TAA)), which has a conserved predicted binding site for AF357425/SNORD113-6. Adjacent to this site, the tRNA is cleaved to form tRFLeu 47-64 in both primary murine and human fibroblasts and in intact human arteries. We show that AF357425/SNORD113-6 methylates tRNALeu(TAA) and thereby prevents the formation of tRFLeu 47-64. Exposing fibroblasts to oxidative or hypoxic stress increased AF357425/SNORD113-6 and tRNALeu(TAA) expression, but AF357425/SNORD113-6 knockdown did not increase tRFLeu 47-64 formation under stress even further. Thus, independent of cellular stress, AF357425/SNORD113-6 protects against site-specific fragmentation of tRNALeu(TAA) via 2'O-ribose-methylation.

BACKGROUND:Pre- and postoperative anxiety is a common phenomenon associated with negative postoperative outcomes. Symptoms of posttraumatic stress disorder, such as fear, nightmares, and sleep deprivation, are prevalent in approximately 30% to 50% of patients following discharge from intensive care units after cardiac surgery. Preliminary evidence suggests a promising role of virtual reality (VR) in preventing stress-related reactions using stress inoculation training. Such training enables cognitive preparation of individuals for stressful situations, thereby becoming more tolerant and resistant to stress, subsequently reducing the risk of potential negative psychological consequences. This study investigated a preoperative VR app-Pre-View-aimed at better informing and preparing patients for cardiac catheterization.OBJECTIVE:This study aims to assess the feasibility, usability, and acceptability of Pre-View in patients undergoing cardiac catheterization.METHODS:Eligible participants were adults scheduled for elective cardiac catheterization. Pre-View comprised an interactive virtual representation of the whole care process related to cardiac catheterization, from entering the hospital for admission to postprocedural stay and discharge. These processes were represented through 360° videos and interactive photos. Self-report questionnaires were completed at baseline (ie, before catheterization and after undergoing the VR experience) and after cardiac catheterization. Outcome measures included user experience and satisfaction, VR presence and immersive tendencies, and user friendliness. The perceived effectiveness was assessed exploratively.RESULTS:A total of 8 individuals, with a mean age of 67 (SD 7.5) years, participated in this study. Half of them underwent the VR experience at the hospital and the other half at home. Participants reported high levels of presence in the virtual environment (Presence Questionnaire score: mean 129.1, SD 13.4). The usability of Pre-View was well evaluated (System Usability Scale score: mean 89.1, SD 12.0), and patient satisfaction was high (Client Satisfaction Questionnaire score: mean 27.1, SD 3.2). Usability and satisfaction scores were higher for participants who underwent Pre-View at home versus those who underwent Pre-View at the hospital, although the latter group was significantly older; 72.8 versus 61.3, respectively. All participants reported Pre-View to be effective in terms of feeling better informed about the care process of cardiac catheterization. Most participants (7/8, 88%) reported Pre-View to be effective in terms of feeling better prepared for cardiac catheterization, acknowledging the potential of Pre-View in reducing negative psychological consequences after catheterization.CONCLUSIONS:The results provide initial support for the feasibility and acceptability of a preoperative VR app, creating a virtual environment that supports patient education and preparation for upcoming cardiac catheterization. More studies are needed to further investigate the effects of VR as a tool to better prepare patients for medical procedures, its effectiveness in reducing negative patient outcomes (eg, anxiety, stress, and postoperative recovery outcomes), and the generalizability of effects across different settings and patient populations.

We have previously shown that treatment with third-generation antisense oligonucleotides against miR-494-3p (3GA-494) reduces atherosclerotic plaque progression and stabilizes lesions, both in early and established plaques, with reduced macrophage content in established plaques. Within the plaque, different subtypes of macrophages are present. Here, we aimed to investigate whether miR-494-3p directly influences macrophage polarization and activation. Human macrophages were polarized into either proinflammatory M1 or anti-inflammatory M2 macrophages and simultaneously treated with 3GA-494 or a control antisense (3GA-ctrl). We show that 3GA-494 treatment inhibited miR-494-3p in M1 macrophages and dampened M1 polarization, while in M2 macrophages miR-494-3p expression was induced and M2 polarization enhanced. The proinflammatory marker CCR2 was reduced in 3GA-494-treated atherosclerosis-prone mice. Pathway enrichment analysis predicted an overlap between miR-494-3p target genes in macrophage polarization and Wnt signaling. We demonstrate that miR-494-3p regulates expression levels of multiple Wnt signaling components, such as LRP6 and TBL1X. Wnt signaling appears activated upon treatment with 3GA-494, both in cultured M1 macrophages and in plaques of hypercholesterolemic mice. Taken together, 3GA-494 treatment dampened M1 polarization, at least in part via activated Wnt signaling, while M2 polarization was enhanced, which is both favorable in reducing atherosclerotic plaque formation and increasing plaque stability.

MicroRNAs are posttranscriptional regulators of gene expression. As microRNAs can target many genes simultaneously, microRNAs can regulate complex multifactorial processes, including post-ischemic neovascularization, a major recovery pathway in cardiovascular disease. MicroRNAs select their target mRNAs via full complementary binding with their seed sequence, i.e., nucleotides 2-8 from the 5' end of a microRNA. The exact sequence of a mature microRNA, and thus of its 5' and 3' ends, is determined by two sequential cleavage steps of microRNA precursors, Drosha/DGCR8 and Dicer. When these cleavage steps result in nucleotide switches at the 5' end, forming a so-called 5'-isomiR, this results in a shift in the mature microRNA's seed sequence. The role of 5'-isomiRs in cardiovascular diseases is still unknown. Here, we characterize the expression and function of the 5'-isomiR of miR-411 (ISO-miR-411). ISO-miR-411 is abundantly expressed in human primary vascular cells. ISO-miR-411 has a different "targetome" from WT-miR-411, with only minor overlap. The ISO-miR-411/WT-miR-411 ratio is downregulated under acute ischemia, both in cells and a murine ischemia model, but is upregulated instead in chronically ischemic human blood vessels. ISO-miR-411 negatively influences vascular cell migration, whereas WT-miR-411 does not. Our data demonstrate that isomiR formation is a functional pathway that is actively regulated during ischemia.