MicroRNAs (miRs) are small, noncoding RNAs that posttranscriptionally control gene expression by inhibiting protein translation or inducing target mRNA destabilization. Besides their intracellular ...function, recent studies demonstrate that miRs can be exported or released by cells and circulate with the blood in a remarkably stable form. The discovery of circulating miRs opens up intriguing possibilities to use the circulating miR patterns as biomarker for cardiovascular diseases. Cardiac injury as it occurs after acute myocardial infarction increases the circulating levels of several myocardial-derived miRs (eg, miR-1, miR-133, miR-499, miR-208), whereas patients with coronary artery disease or diabetes showed reduced levels of endothelial-enriched miRs, such as miR-126. This review article summarizes the current clinical and experimental studies addressing the role of circulating miRs as a diagnostic or prognostic biomarker in cardiovascular disease. In addition, the mechanisms by which miRs are released and their putative function as long-distance communicators are discussed.
Coronavirus disease 2019 (COVID-19) continues to cause considerable morbidity and mortality worldwide. Case reports of hospitalized patients suggest that COVID-19 prominently affects the ...cardiovascular system, but the overall impact remains unknown.
To evaluate the presence of myocardial injury in unselected patients recently recovered from COVID-19 illness.
In this prospective observational cohort study, 100 patients recently recovered from COVID-19 illness were identified from the University Hospital Frankfurt COVID-19 Registry between April and June 2020.
Recent recovery from severe acute respiratory syndrome coronavirus 2 infection, as determined by reverse transcription-polymerase chain reaction on swab test of the upper respiratory tract.
Demographic characteristics, cardiac blood markers, and cardiovascular magnetic resonance (CMR) imaging were obtained. Comparisons were made with age-matched and sex-matched control groups of healthy volunteers (n = 50) and risk factor-matched patients (n = 57).
Of the 100 included patients, 53 (53%) were male, and the mean (SD) age was 49 (14) years. The median (IQR) time interval between COVID-19 diagnosis and CMR was 71 (64-92) days. Of the 100 patients recently recovered from COVID-19, 67 (67%) recovered at home, while 33 (33%) required hospitalization. At the time of CMR, high-sensitivity troponin T (hsTnT) was detectable (greater than 3 pg/mL) in 71 patients recently recovered from COVID-19 (71%) and significantly elevated (greater than 13.9 pg/mL) in 5 patients (5%). Compared with healthy controls and risk factor-matched controls, patients recently recovered from COVID-19 had lower left ventricular ejection fraction, higher left ventricle volumes, and raised native T1 and T2. A total of 78 patients recently recovered from COVID-19 (78%) had abnormal CMR findings, including raised myocardial native T1 (n = 73), raised myocardial native T2 (n = 60), myocardial late gadolinium enhancement (n = 32), or pericardial enhancement (n = 22). There was a small but significant difference between patients who recovered at home vs in the hospital for native T1 mapping (median IQR, 1119 1092-1150 ms vs 1141 1121-1175 ms; P = .008) and hsTnT (4.2 3.0-5.9 pg/dL vs 6.3 3.4-7.9 pg/dL; P = .002) but not for native T2 mapping. None of these measures were correlated with time from COVID-19 diagnosis (native T1: r = 0.07; P = .47; native T2: r = 0.14; P = .15; hsTnT: r = -0.07; P = .50). High-sensitivity troponin T was significantly correlated with native T1 mapping (r = 0.33; P < .001) and native T2 mapping (r = 0.18; P = .01). Endomyocardial biopsy in patients with severe findings revealed active lymphocytic inflammation. Native T1 and T2 were the measures with the best discriminatory ability to detect COVID-19-related myocardial pathology.
In this study of a cohort of German patients recently recovered from COVID-19 infection, CMR revealed cardiac involvement in 78 patients (78%) and ongoing myocardial inflammation in 60 patients (60%), independent of preexisting conditions, severity and overall course of the acute illness, and time from the original diagnosis. These findings indicate the need for ongoing investigation of the long-term cardiovascular consequences of COVID-19.
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.
Recent statistics indicate that the human population is ageing rapidly. Healthy, but also diseased, elderly people are increasing. This trend is particularly evident in Western countries, where ...healthier living conditions and better cures are available. To understand the process leading to age-associated alterations is, therefore, of the highest relevance for the development of new treatments for age-associated diseases, such as cancer, diabetes, Alzheimer and cardiovascular accidents. Mechanistically, it is well accepted that the accumulation of intracellular damage determined by reactive oxygen species (ROS) might orchestrate the progressive loss of control over biological homeostasis and the functional impairment typical of aged tissues. Here, we review how epigenetics takes part in the control of stress stimuli and the mechanisms of ageing physiology and physiopathology. Alteration of epigenetic enzyme activity, histone modifications and DNA-methylation is, in fact, typically associated with the ageing process. Specifically, ageing presents peculiar epigenetic markers that, taken altogether, form the still ill-defined "ageing epigenome". The comprehension of mechanisms and pathways leading to epigenetic modifications associated with ageing may help the development of anti-ageing therapies.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Clonal hematopoiesis driven by mutations of DNMT3A (DNA methyltransferase 3a) is associated with increased incidence of cardiovascular disease and poor prognosis of patients with chronic heart ...failure (HF) and aortic stenosis. Although experimental studies suggest that DNMT3A clonal hematopoiesis-driver mutations may enhance inflammation, specific signatures of inflammatory cells in humans are missing.
To define subsets of immune cells mediating inflammation in humans using single-cell RNA sequencing.
Transcriptomic profiles of peripheral blood mononuclear cells were analyzed in n=6 patients with HF harboring DNMT3A clonal hematopoiesis-driver mutations and n=4 patients with HF and no DNMT3A mutations by single-cell RNA sequencing. Monocytes of patients with HF carrying DNMT3A mutations demonstrated a significantly increased expression of inflammatory genes compared with monocytes derived from patients with HF without DNMT3A mutations. Among the specific upregulated genes were the prototypic inflammatory IL (interleukin)
, the inflammasome
, and the macrophage inflammatory proteins
and
as well as resistin, which augments monocyte-endothelial adhesion. Silencing of DNMT3A in monocytes induced a paracrine proinflammatory activation and increased adhesion to endothelial cells. Furthermore, the classical monocyte subset of DNMT3A mutation carriers showed increased expression of T-cell stimulating immunoglobulin superfamily members
,
,
, as well as the CD2 ligand and cell adhesion molecule
, all of which may be involved in monocyte-T-cell interactions. DNMT3A mutation carriers were further characterized by increased expression of the T-cell alpha receptor constant chain and changes in T helper cell 1, T helper cell 2, T helper cell 17, CD8+ effector, CD4+ memory, and regulatory T-cell-specific signatures.
This study demonstrates that circulating monocytes and T cells of patients with HF harboring clonal hematopoiesis-driver mutations in DNMT3A exhibit a highly inflamed transcriptome, which may contribute to the aggravation of chronic HF.
Abstract
Aims
Clonal haematopoiesis of indeterminate potential (CHIP), defined as the presence of an expanded somatic blood cell clone without other haematological abnormalities, was recently shown ...to increase with age and is associated with coronary artery disease and calcification. The most commonly mutated CHIP genes, DNMT3A and TET2, were shown to regulate inflammatory potential of circulating leucocytes. The incidence of degenerative calcified aortic valve (AV) stenosis increases with age and correlates with chronic inflammation. We assessed the incidence of CHIP and its association with inflammatory blood cell phenotypes in patients with AV stenosis undergoing transfemoral aortic valve implantation (TAVI).
Methods and results
Targeted amplicon sequencing for DNMT3A and TET2 was performed in 279 patients with severe AV stenosis undergoing TAVI. Somatic DNMT3A- or TET2-CHIP-driver mutations with a VAF ≥ 2% were detected in 93 out of 279 patients (33.3%), with an age-dependent increase in the incidence from 25% (55–69 years) to 52.9% (90–100 years). Patients with DNMT3A- or TET2-CHIP-driver mutations did not differ from patients without such mutations in clinical parameters, concomitant atherosclerotic disease, blood cell counts, inflammatory markers, or procedural characteristics. However, patients with DNMT3A- or TET2-CHIP-driver mutations had a profoundly increased medium-term all-cause mortality following successful TAVI. Differential myeloid and T-cell distributions revealed pro-inflammatory T-cell polarization in DNMT3A-mutation carriers and increased pro-inflammatory non-classical monocytes in TET2-mutation carriers.
Conclusion
This is the first study to show that acquired somatic mutations in the most commonly mutated CHIP-driver genes occur frequently in patients with severe degenerative AV stenosis, are associated with increased pro-inflammatory leucocyte subsets, and confer a profound increase in mortality following successful TAVI.
Abstract
Aims
Coronavirus disease 2019 is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and has emerged as a global pandemic. SARS-CoV-2 infection can lead to elevated ...markers of cardiac injury associated with higher risk of mortality. It is unclear whether cardiac injury is caused by direct infection of cardiomyocytes or is mainly secondary to lung injury and inflammation. Here, we investigate whether cardiomyocytes are permissive for SARS-CoV-2 infection.
Methods and results
Two strains of SARS-CoV-2 infected human induced pluripotent stem cell-derived cardiomyocytes as demonstrated by detection of intracellular double-stranded viral RNA and viral spike glycoprotein expression. Increasing concentrations of viral RNA are detected in supernatants of infected cardiomyocytes, which induced infections in Caco-2 cell lines, documenting productive infections. SARS-CoV-2 infection and induced cytotoxic and proapoptotic effects associated with it abolished cardiomyocyte beating. RNA sequencing confirmed a transcriptional response to viral infection as demonstrated by the up-regulation of genes associated with pathways related to viral response and interferon signalling, apoptosis, and reactive oxygen stress. SARS-CoV-2 infection and cardiotoxicity was confirmed in a 3D cardiosphere tissue model. Importantly, viral spike protein and viral particles were detected in living human heart slices after infection with SARS-CoV-2. Coronavirus particles were further observed in cardiomyocytes of a patient with coronavirus disease 2019. Infection of induced pluripotent stem cell-derived cardiomyocytes was dependent on cathepsins and angiotensin-converting enzyme 2, and was blocked by remdesivir.
Conclusion
This study demonstrates that SARS-CoV-2 infects cardiomyocytes in vitro in an angiotensin-converting enzyme 2- and cathepsin-dependent manner. SARS-CoV-2 infection of cardiomyocytes is inhibited by the antiviral drug remdesivir.
Graphical Abstract
MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression by binding to the cellular transcript leading to translational repression or degradation of the target mRNA. Dicer and Drosha ...are the miRNA processing enzymes that are required for the maturation of miRNAs. Here, we investigated the role of Dicer and Drosha for angiogenesis. Endothelial cells were transfected with siRNA against Dicer and Drosha to inhibit miRNA biogenesis. Genetic silencing of Dicer and Drosha significantly reduced capillary sprouting of endothelial cells and tube forming activity. Migration of endothelial cells was significantly decreased in Dicer siRNA–transfected cells, whereas Drosha siRNA had no effect. Silencing of Dicer but not of Drosha reduced angiogenesis in vivo. Next, we attempted to identify miRNAs expressed in endothelial cells. A screening analysis of 168 human miRNAs using real-time PCR revealed that members of the let-7 family, mir-21, mir-126, mir-221, and mir-222 are highly expressed in endothelial cells. Dicer and Drosha siRNA reduced lef-7f and mir-27b expression. Inhibitors against let-7f and mir-27b also reduced sprout formation indicating that let-7f and mir-27b promote angiogenesis by targeting antiangiogenic genes. In silico analysis of predicted targets for let-7 cluster identified the endogenous angiogenesis inhibitor thrombospondin-1. Indeed, Dicer and Drosha siRNA significantly increased the expression of thrombospondin-1. Taken together, transient reduction of the miRNA-regulating enzyme Dicer impairs angiogenesis in vitro and in vivo, whereas Drosha siRNA induced a minor antiangiogenic effect in vitro and was not effective in vivo. The let-7 family and mir-27b appear to be attractive targets for modulating angiogenesis.
Circulating endothelial progenitor cells (EPC), involved in endothelial regeneration, neovascularisation, and determination of prognosis in cardiovascular disease can be characterised with functional ...assays or using immunofluorescence and flow cytometry. Combinations of markers, including CD34+KDR+ or CD133+KDR+, are used. This approach, however may not consider all characteristics of EPC. The lack of a standardised protocol with regards to reagents and gating strategies may account for the widespread inter-laboratory variations in quantification of EPC. We, therefore developed a novel protocol adapted from the standardised so-called ISHAGE protocol for enumeration of haematopoietic stem cells to enable comparison of clinical and laboratory data.
In 25 control subjects, 65 patients with coronary artery disease (CAD; 40 stable CAD, 25 acute coronary syndrome/acute myocardial infarction (ACS)), EPC were quantified using the following approach: Whole blood was incubated with CD45, KDR, and CD34. The ISHAGE sequential strategy was used, and finally, CD45(dim)CD34(+) cells were quantified for KDR. A minimum of 100 CD34(+) events were collected. For comparison, CD45(+)CD34(+) and CD45(-)CD34(+) were analysed simultaneously. The number of CD45(dim)CD34(+)KDR(+) cells only were significantly higher in healthy controls compared to patients with CAD or ACS (p = 0.005 each, p<0.001 for trend). An inverse correlation of CD45(dim)CD34(+)KDR(+) with disease activity (r = -0.475, p<0.001) was confirmed. Only CD45(dim)CD34(+)KDR(+) correlated inversely with the number of diseased coronaries (r = -0.344; p<0.005). In a second study, a 4-week de-novo treatment of atorvastatin in stable CAD evoked an increase only of CD45(dim)CD34(+)KDR(+) EPC (p<0.05). CD45(+)CD34(+)KDR(+) and CD45(-)CD34(+)KDR(+) were indifferent between the three groups.
Our newly established protocol adopted from the standardised ISHAGE protocol achieved higher accuracy in EPC enumeration confirming previous findings with respect to the correlation of EPC with disease activity and the increase of EPC during statin therapy. The data of this study show the CD45(dim) fraction to harbour EPC.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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