miR-210 as a therapeutic target in diabetes-associated endothelial dysfunction.

BACKGROUND AND PURPOSE: MicroRNA (miR)-210 function in endothelial cells and its role in diabetes-associated endothelial dysfunction are not fully understood. We aimed to characterize the miR-210 function in endothelial cells and study its therapeutic potential in diabetes. EXPERIMENTAL APPROACH: Two different diabetic mouse models (db/db and Western diet-induced), miR-210 knockout and transgenic mice, isolated vessels and human endothelial cells were used. KEY RESULTS: miR-210 levels were lower in aortas isolated from db/db than in control mice. Endothelium-dependent relaxation (EDR) was impaired in aortas from miR-210 knockout mice, and this was restored by inhibiting miR-210 downstream protein tyrosine phosphatase 1B (PTP1B), mitochondrial glycerol-3-phosphate dehydrogenase 2 (GPD2), and mitochondrial oxidative stress. Inhibition of these pathways also improved EDR in both diabetic mouse models. High glucose reduced miR-210 levels in endothelial cells and impaired EDR in mouse aortas, effects that were reversed by overexpressing miR-210. However, plasma miR-210 levels were not affected in individuals with type 2 diabetes (T2D) following improved glycaemic status. Of note, genetic overexpression using miR-210 transgenic mice and pharmacological overexpression using miR-210 mimic in vivo ameliorated endothelial dysfunction in both diabetic mouse models by decreasing PTP1B, GPD2 and oxidative stress. Genetic overexpression of miR-210 altered the aortic transcriptome, decreasing genes in pathways involved in oxidative stress. miR-210 mimic restored decreased nitric oxide production by high glucose in endothelial cells. CONCLUSION AND IMPLICATIONS: This study unravels the mechanisms by which down-regulated miR-210 by high glucose induces endothelial dysfunction in T2D and demonstrates that miR-210 serves as a novel therapeutic target.

miR-210 is essential to retinal homeostasis in fruit flies and mice.

BACKGROUND: miR-210 is one of the most evolutionarily conserved microRNAs. It is known to be involved in several physiological and pathological processes, including response to hypoxia, angiogenesis, cardiovascular diseases and cancer. Recently, new roles of this microRNA are emerging in the context of eye and visual system homeostasis. Recent studies in Drosophila melanogaster unveiled that the absence of miR-210 leads to a progressive retinal degeneration characterized by the accumulation of lipid droplets and disruptions in lipid metabolism. However, the possible conservation of miR-210 knock-out effect in the mammalian retina has yet to be explored. RESULTS: We further investigated lipid anabolism and catabolism in miR-210 knock-out (KO) flies, uncovering significant alterations in gene expression within these pathways. Additionally, we characterized the retinal morphology of flies overexpressing (OE) miR-210, which was not affected by the increased levels of the microRNA. For the first time, we also characterized the retinal morphology of miR-210 KO and OE mice. Similar to flies, miR-210 OE did not affect retinal homeostasis, whereas miR-210 KO mice exhibited photoreceptor degeneration. To explore other potential parallels between miR-210 KO models in flies and mice, we examined lipid metabolism, circadian behaviour, and retinal transcriptome in mice, but found no similarities. Specifically, RNA-seq confirmed the lack of involvement of lipid metabolism in the mice's pathological phenotype, revealing that the differentially expressed genes were predominantly associated with chloride channel activity and extracellular matrix homeostasis. Simultaneously, transcriptome analysis of miR-210 KO fly brains indicated that the observed alterations extend beyond the eye and may be linked to neuronal deficiencies in signal detection and transduction. CONCLUSIONS: We provide the first morphological characterization of the retina of miR-210 KO and OE mice, investigating the role of this microRNA in mammalian retinal physiology and exploring potential parallels with phenotypes observed in fly models. Although the lack of similarities in lipid metabolism, circadian behaviour, and retinal transcriptome in mice suggests divergent mechanisms of retinal degeneration between the two species, transcriptome analysis of miR-210 KO fly brains indicates the potential existence of a shared upstream mechanism contributing to retinal degeneration in both flies and mammals.

Coding and Non-Coding Transcriptomic Landscape of Aortic Complications in Marfan Syndrome.

Marfan syndrome (MFS) is a rare congenital disorder of the connective tissue, leading to thoracic aortic aneurysms (TAA) and dissection, among other complications. Currently, the most efficient strategy to prevent life-threatening dissection is preventive surgery. Periodic imaging applying complex techniques is required to monitor TAA progression and to guide the timing of surgical intervention. Thus, there is an acute demand for non-invasive biomarkers for diagnosis and prognosis, as well as for innovative therapeutic targets of MFS. Unraveling the intricate pathomolecular mechanisms underlying the syndrome is vital to address these needs. High-throughput platforms are particularly well-suited for this purpose, as they enable the integration of different datasets, such as transcriptomic and epigenetic profiles. In this narrative review, we summarize relevant studies investigating changes in both the coding and non-coding transcriptome and epigenome in MFS-induced TAA. The collective findings highlight the implicated pathways, such as TGF-ß signaling, extracellular matrix structure, inflammation, and mitochondrial dysfunction. Potential candidates as biomarkers, such as miR-200c, as well as therapeutic targets emerged, like Tfam, associated with mitochondrial respiration, or miR-632, stimulating endothelial-to-mesenchymal transition. While these discoveries are promising, rigorous and extensive validation in large patient cohorts is indispensable to confirm their clinical relevance and therapeutic potential.

Circular RNA regulatory role in pathological cardiac remodelling.

Cardiac remodelling involves structural, cellular and molecular alterations in the heart after injury, resulting in progressive loss of heart function and ultimately leading to heart failure. Circular RNAs (circRNAs) are a recently rediscovered class of non-coding RNAs that play regulatory roles in the pathogenesis of cardiovascular diseases, including heart failure. Thus, a more comprehensive understanding of the role of circRNAs in the processes governing cardiac remodelling may set the ground for the development of circRNA-based diagnostic and therapeutic strategies. In this review, the current knowledge about circRNA origin, conservation, characteristics and function is summarized. Bioinformatics and wet-lab methods used in circRNA research are discussed. The regulatory function of circRNAs in cardiac remodelling mechanisms such as cell death, cardiomyocyte hypertrophy, inflammation, fibrosis and metabolism is highlighted. Finally, key challenges and opportunities in circRNA research are discussed, and orientations for future work to address the pharmacological potential of circRNAs in heart failure are proposed.

Development of a long noncoding RNA-based machine learning model to predict COVID-19 in-hospital mortality.

Tools for predicting COVID-19 outcomes enable personalized healthcare, potentially easing the disease burden. This collaborative study by 15 institutions across Europe aimed to develop a machine learning model for predicting the risk of in-hospital mortality post-SARS-CoV-2 infection. Blood samples and clinical data from 1286 COVID-19 patients collected from 2020 to 2023 across four cohorts in Europe and Canada were analyzed, with 2906 long non-coding RNAs profiled using targeted sequencing. From a discovery cohort combining three European cohorts and 804 patients, age and the long non-coding RNA LEF1-AS1 were identified as predictive features, yielding an AUC of 0.83 (95% CI 0.82-0.84) and a balanced accuracy of 0.78 (95% CI 0.77-0.79) with a feedforward neural network classifier. Validation in an independent Canadian cohort of 482 patients showed consistent performance. Cox regression analysis indicated that higher levels of LEF1-AS1 correlated with reduced mortality risk (age-adjusted hazard ratio 0.54, 95% CI 0.40-0.74). Quantitative PCR validated LEF1-AS1's adaptability to be measured in hospital settings. Here, we demonstrate a promising predictive model for enhancing COVID-19 patient management.

Addressing the unsolved challenges in microRNA-based biomarker development: Suitable endogenous reference microRNAs for SARS-CoV-2 infection severity.

Circulating cell-free microRNAs (miRNAs) are promising biomarkers for medical decision-making. Suitable endogenous controls are essential to ensure reproducibility. We aimed to identify and validate endogenous reference miRNAs for qPCR data normalization in samples from SARS-CoV-2-infected hospitalized patients. We used plasma samples (n = 170) from COVID-19 patients collected at hospital admission (COVID-Ponent project, www.clinicaltrials.gov/NCT04824677). First, 179 miRNAs were profiled using RT-qPCR. After stability assessment, candidates were validated using the same methodology. miRNA stability was analyzed using the geNorm, NormFinder and BestKeeper algorithms. Stability was further evaluated using an RNA-seq dataset derived from COVID-19 hospitalized patients, along with plasma samples from patients with critical COVID-19 profiled using RT-qPCR. In the screening phase, after strict control of expression levels, stability assessment selected eleven candidates (miR-17-5p, miR-20a-5p, miR-30e-5p, miR-106a-5p, miR-151a-5p, miR-185-5p, miR-191-5p, miR-423-3p, miR-425-5p, miR-484 and miR-625-5p). In the validation phase, all algorithms identified miR-106a-5p and miR-484 as top endogenous controls. No association was observed between these miRNAs and clinical or sociodemographic characteristics. Both miRNAs were stably detected and showed low variability in the additional analyses. In conclusion, a 2-miRNA panel composed of miR-106a-5p and miR-484 constitutes a first-line normalizer for miRNA-based biomarker development using qPCR in hospitalized patients infected with SARS-CoV-2.

Non-coding RNAs as therapeutic targets and biomarkers in ischaemic heart disease.

The adult heart is a complex, multicellular organ that is subjected to a series of regulatory stimuli and circuits and has poor reparative potential. Despite progress in our understanding of disease mechanisms and in the quality of health care, ischaemic heart disease remains the leading cause of death globally, owing to adverse cardiac remodelling, leading to ischaemic cardiomyopathy and heart failure. Therapeutic targets are urgently required for the protection and repair of the ischaemic heart. Moreover, personalized clinical biomarkers are necessary for clinical diagnosis, medical management and to inform the individual response to treatment. Non-coding RNAs (ncRNAs) deeply influence cardiovascular functions and contribute to communication between cells in the cardiac microenvironment and between the heart and other organs. As such, ncRNAs are candidates for translation into clinical practice. However, ncRNA biology has not yet been completely deciphered, given that classes and modes of action have emerged only in the past 5 years. In this Review, we discuss the latest discoveries from basic research on ncRNAs and highlight both the clinical value and the challenges underscoring the translation of these molecules as biomarkers and therapeutic regulators of the processes contributing to the initiation, progression and potentially the prevention or resolution of ischaemic heart disease and heart failure.

Recommendations for detection, validation, and evaluation of RNA editing events in cardiovascular and neurological/neurodegenerative diseases.

RNA editing, a common and potentially highly functional form of RNA modification, encompasses two different RNA modifications, namely adenosine to inosine (A-to-I) and cytidine to uridine (C-to-U) editing. As inosines are interpreted as guanosines by the cellular machinery, both A-to-I and C-to-U editing change the nucleotide sequence of the RNA. Editing events in coding sequences have the potential to change the amino acid sequence of proteins, whereas editing events in noncoding RNAs can, for example, affect microRNA target binding. With advancing RNA sequencing technology, more RNA editing events are being discovered, studied, and reported. However, RNA editing events are still often overlooked or discarded as sequence read quality defects. With this position paper, we aim to provide guidelines and recommendations for the detection, validation, and follow-up experiments to study RNA editing, taking examples from the fields of cardiovascular and brain disease. We discuss all steps, from sample collection, storage, and preparation, to different strategies for RNA sequencing and editing-sensitive data analysis strategies, to validation and follow-up experiments, as well as potential pitfalls and gaps in the available technologies. This paper may be used as an experimental guideline for RNA editing studies in any disease context.

circRNA-miRNA-mRNA Deregulated Network in Ischemic Heart Failure Patients.

Noncoding RNAs (ncRNAs), which include circular RNAs (circRNAs) and microRNAs (miRNAs), regulate the development of cardiovascular diseases (CVD). Notably, circRNAs can interact with miRNAs, influencing their specific mRNA targets' levels and shaping a competing endogenous RNAs (ceRNA) network. However, these interactions and their respective functions remain largely unexplored in ischemic heart failure (IHF). This study is aimed at identifying circRNA-centered ceRNA networks in non-end-stage IHF. Approximately 662 circRNA-miRNA-mRNA interactions were identified in the heart by combining state-of-the-art bioinformatics tools with experimental data. Importantly, KEGG terms of the enriched mRNA indicated CVD-related signaling pathways. A specific network centered on circBPTF was validated experimentally. The levels of let-7a-5p, miR-18a-3p, miR-146b-5p, and miR-196b-5p were enriched in circBPTF pull-down experiments, and circBPTF silencing inhibited the expression of HDAC9 and LRRC17, which are targets of miR-196b-5p. Furthermore, as suggested by the enriched pathway terms of the circBPTF ceRNA network, circBPTF inhibition elicited endothelial cell cycle arrest. circBPTF expression increased in endothelial cells exposed to hypoxia, and its upregulation was confirmed in cardiac samples of 36 end-stage IHF patients compared to healthy controls. In conclusion, circRNAs act as miRNA sponges, regulating the functions of multiple mRNA targets, thus providing a novel vision of HF pathogenesis and laying the theoretical foundation for further experimental studies.

HCG18, LEF1AS1 and lncCEACAM21 as biomarkers of disease severity in the peripheral blood mononuclear cells of COVID-19 patients.

BACKGROUND: Even after 3 years from SARS-CoV-2 identification, COVID-19 is still a persistent and dangerous global infectious disease. Significant improvements in our understanding of the disease pathophysiology have now been achieved. Nonetheless, reliable and accurate biomarkers for the early stratification of COVID-19 severity are still lacking. Long noncoding RNAs (LncRNAs) are ncRNAs longer than 200 nucleotides, regulating the transcription and translation of protein-coding genes and they can be found in the peripheral blood, thus holding a promising biomarker potential. Specifically, peripheral blood mononuclear cells (PBMCs) have emerged as a source of indirect biomarkers mirroring the conditions of tissues: they include monocytes, B and T lymphocytes, and natural killer T cells (NKT), being highly informative for immune-related events. METHODS: We profiled by RNA-Sequencing a panel of 2906 lncRNAs to investigate their modulation in PBMCs of a pilot group of COVID-19 patients, followed by qPCR validation in 111 hospitalized COVID-19 patients. RESULTS: The levels of four lncRNAs were found to be decreased in association with COVID-19 mortality and disease severity: HLA Complex Group 18-242 and -244 (HCG18-242 and HCG18-244), Lymphoid Enhancer Binding Factor 1-antisense 1 (LEF1-AS1) and lncCEACAM21 (i.e. ENST00000601116.5, a lncRNA in the CEACAM21 locus). Interestingly, these deregulations were confirmed in an independent patient group of hospitalized patients and by the re-analysis of publicly available single-cell transcriptome datasets. The identified lncRNAs were expressed in all of the PBMC cell types and inversely correlated with the neutrophil/lymphocyte ratio (NLR), an inflammatory marker. In vitro, the expression of LEF1-AS1 and lncCEACAM21 was decreased upon THP-1 monocytes exposure to a relevant stimulus, hypoxia. CONCLUSION: The identified COVID-19-lncRNAs are proposed as potential innovative biomarkers of COVID-19 severity and mortality.

Integration of epigenetic regulatory mechanisms in heart failure.

The number of "omics" approaches is continuously growing. Among others, epigenetics has appeared as an attractive area of investigation by the cardiovascular research community, notably considering its association with disease development. Complex diseases such as cardiovascular diseases have to be tackled using methods integrating different omics levels, so called "multi-omics" approaches. These approaches combine and co-analyze different levels of disease regulation. In this review, we present and discuss the role of epigenetic mechanisms in regulating gene expression and provide an integrated view of how these mechanisms are interlinked and regulate the development of cardiac disease, with a particular attention to heart failure. We focus on DNA, histone, and RNA modifications, and discuss the current methods and tools used for data integration and analysis. Enhancing the knowledge of these regulatory mechanisms may lead to novel therapeutic approaches and biomarkers for precision healthcare and improved clinical outcomes.

Cardiovascular complications of diabetes: role of non-coding RNAs in the crosstalk between immune and cardiovascular systems.

Diabetes mellitus, a group of metabolic disorders characterized by high levels of blood glucose caused by insulin defect or impairment, is a major risk factor for cardiovascular diseases and related mortality. Patients with diabetes experience a state of chronic or intermittent hyperglycemia resulting in damage to the vasculature, leading to micro- and macro-vascular diseases. These conditions are associated with low-grade chronic inflammation and accelerated atherosclerosis. Several classes of leukocytes have been implicated in diabetic cardiovascular impairment. Although the molecular pathways through which diabetes elicits an inflammatory response have attracted significant attention, how they contribute to altering cardiovascular homeostasis is still incompletely understood. In this respect, non-coding RNAs (ncRNAs) are a still largely under-investigated class of transcripts that may play a fundamental role. This review article gathers the current knowledge on the function of ncRNAs in the crosstalk between immune and cardiovascular cells in the context of diabetic complications, highlighting the influence of biological sex in such mechanisms and exploring the potential role of ncRNAs as biomarkers and targets for treatments. The discussion closes by offering an overview of the ncRNAs involved in the increased cardiovascular risk suffered by patients with diabetes facing Sars-CoV-2 infection.

The science behind soft skills: Do's and Don'ts for early career researchers and beyond. A review paper from the EU-CardioRNA COST Action CA17129.

Soft skills are the elementary management, personal, and interpersonal abilities that are vital for an individual to be efficient at workplace or in their personal life. Each work place requires different set of soft skills. Thus, in addition to scientific/technical skills that are easier to access within a short time frame, several key soft skills are essential for the success of a researcher in today's international work environment. In this paper, the trainees and trainers of the EU-CardioRNA COST Action CA17129 training school on soft skills present basic and advanced soft skills for early career researchers. Here, we particularly emphasize on the importance of transferable and presentation skills, ethics, literature reading and reviewing, research protocol and grant writing, networking, and career opportunities for researchers. All these skills are vital but are often overlooked by some scholars. We also provide tips to ace in aforementioned skills that are crucial in a day-to-day life of early and late career researchers in academia and industry.

Association of miR-144 levels in the peripheral blood with COVID-19 severity and mortality.

Coronavirus disease-2019 (COVID-19) can be asymptomatic or lead to a wide symptom spectrum, including multi-organ damage and death. Here, we explored the potential of microRNAs in delineating patient condition and predicting clinical outcome. Plasma microRNA profiling of hospitalized COVID-19 patients showed that miR-144-3p was dynamically regulated in response to COVID-19. Thus, we further investigated the biomarker potential of miR-144-3p measured at admission in 179 COVID-19 patients and 29 healthy controls recruited in three centers. In hospitalized patients, circulating miR-144-3p levels discriminated between non-critical and critical illness (AUCmiR-144-3p = 0.71; p = 0.0006), acting also as mortality predictor (AUCmiR-144-3p = 0.67; p = 0.004). In non-hospitalized patients, plasma miR-144-3p levels discriminated mild from moderate disease (AUCmiR-144-3p = 0.67; p = 0.03). Uncontrolled release of pro-inflammatory cytokines can lead to clinical deterioration. Thus, we explored the added value of a miR-144/cytokine combined analysis in the assessment of hospitalized COVID-19 patients. A miR-144-3p/Epidermal Growth Factor (EGF) combined score discriminated between non-critical and critical hospitalized patients (AUCmiR-144-3p/EGF = 0.81; p < 0.0001); moreover, a miR-144-3p/Interleukin-10 (IL-10) score discriminated survivors from nonsurvivors (AUCmiR-144-3p/IL-10 = 0.83; p < 0.0001). In conclusion, circulating miR-144-3p, possibly in combination with IL-10 or EGF, emerges as a noninvasive tool for early risk-based stratification and mortality prediction in COVID-19.

CircANKRD12 Is Induced in Endothelial Cell Response to Oxidative Stress.

Redox imbalance of the endothelial cells (ECs) plays a causative role in a variety of cardiovascular diseases. In order to better understand the molecular mechanisms of the endothelial response to oxidative stress, the involvement of circular RNAs (circRNAs) was investigated. CircRNAs are RNA species generated by a "back-splicing" event, which is the covalent linking of the 3'- and 5'-ends of exons. Bioinformatics analysis of the transcriptomic landscape of human ECs exposed to H2O2 allowed us to identify a subset of highly expressed circRNAs compared to their linear RNA counterparts, suggesting a potential biological relevance. Specifically, circular Ankyrin Repeat Domain 12 (circANKRD12), derived from the junction of exon 2 and exon 8 of the ANKRD12 gene (hsa_circ_0000826), was significantly induced in H2O2-treated ECs. Conversely, the linear RNA isoform of ANKRD12 was not modulated. An increased circular-to-linear ratio of ANKRD12 was also observed in cultured ECs exposed to hypoxia and in skeletal muscle biopsies of patients affected by critical limb ischemia (CLI), two conditions associated with redox imbalance and oxidative stress. The functional relevance of circANKRD12 was shown by the inhibition of EC formation of capillary-like structures upon silencing of the circular but not of the linear isoform of ANKRD12. Bioinformatics analysis of the circANKRD12-miRNA-mRNA regulatory network in H2O2-treated ECs identified the enrichment of the p53 and Foxo signaling pathways, both crucial in the cellular response to redox imbalance. In keeping with the antiproliferative action of the p53 pathway, circANKRD12 silencing inhibited EC proliferation. In conclusion, this study indicates circANKRD12 as an important player in ECs exposed to oxidative stress.

Beta-Secretase-1 Antisense RNA Is Associated with Vascular Ageing and Atherosclerotic Cardiovascular Disease.

BACKGROUND: The noncoding antisense transcript for ß-secretase-1 (BACE1-AS) is a long noncoding RNA with a pivotal role in the regulation of amyloid-ß (Aß). We aimed to explore the clinical value of BACE1-AS expression in atherosclerotic cardiovascular disease (ASCVD). METHODS: Expression of BACE1-AS and its target, ß-secretase 1 (BACE1) mRNA, was measured in peripheral blood mononuclear cells derived from 434 individuals (259 without established ASCVD [non-CVD], 90 with stable coronary artery disease [CAD], and 85 with acute coronary syndrome). Intima-media thickness and atheromatous plaques evaluated by ultrasonography, as well as arterial wave reflections and pulse wave velocity, were measured as markers of subclinical ASCVD. Patients were followed for a median of 52 months for major adverse cardiovascular events (MACE). RESULTS: In the cross-sectional arm, BACE1-AS expression correlated with BACE1 expression (r = 0.396, p < 0.001) and marginally with Aß1-40 levels in plasma (r = 0.141, p = 0.008). Higher BACE1-AS was associated with higher estimated CVD risk assessed by HeartScore for non-CVD subjects and by European Society of Cardiology clinical criteria for the total population (p < 0.05 for both). BACE1-AS was associated with higher prevalence of CAD (odds ratio [OR] = 1.85, 95% confidence interval [CI]: 1.37-2.5), multivessel CAD (OR = 1.36, 95% CI: 1.06-1.75), and with higher number of diseased vascular beds (OR = 1.31, 95% CI: 1.07-1.61, for multiple diseased vascular beds) after multivariable adjustment for traditional cardiovascular risk factors. In the prospective arm, BACE1-AS was an independent predictor of MACE in high cardiovascular risk patients (adjusted hazard ratio = 1.86 per ascending tertile, 95% CI: 1.011-3.43, p = 0.046). CONCLUSION: BACE1-AS is associated with the incidence and severity of ASCVD.