LATS1 controls CTCF chromatin occupancy and hormonal response of 3D-grown breast cancer cells.

The cancer epigenome has been studied in cells cultured in two-dimensional (2D) monolayers, but recent studies highlight the impact of the extracellular matrix and the three-dimensional (3D) environment on multiple cellular functions. Here, we report the physical, biochemical, and genomic differences between T47D breast cancer cells cultured in 2D and as 3D spheroids. Cells within 3D spheroids exhibit a rounder nucleus with less accessible, more compacted chromatin, as well as altered expression of ~2000 genes, the majority of which become repressed. Hi-C analysis reveals that cells in 3D are enriched for regions belonging to the B compartment, have decreased chromatin-bound CTCF and increased fusion of topologically associating domains (TADs). Upregulation of the Hippo pathway in 3D spheroids results in the activation of the LATS1 kinase, which promotes phosphorylation and displacement of CTCF from DNA, thereby likely causing the observed TAD fusions. 3D cells show higher chromatin binding of progesterone receptor (PR), leading to an increase in the number of hormone-regulated genes. This effect is in part mediated by LATS1 activation, which favors cytoplasmic retention of YAP and CTCF removal.

TFIIIC Binding to Alu Elements Controls Gene Expression via Chromatin Looping and Histone Acetylation.

How repetitive elements, epigenetic modifications, and architectural proteins interact ensuring proper genome expression remains poorly understood. Here, we report regulatory mechanisms unveiling a central role of Alu elements (AEs) and RNA polymerase III transcription factor C (TFIIIC) in structurally and functionally modulating the genome via chromatin looping and histone acetylation. Upon serum deprivation, a subset of AEs pre-marked by the activity-dependent neuroprotector homeobox Protein (ADNP) and located near cell-cycle genes recruits TFIIIC, which alters their chromatin accessibility by direct acetylation of histone H3 lysine-18 (H3K18). This facilitates the contacts of AEs with distant CTCF sites near promoter of other cell-cycle genes, which also become hyperacetylated at H3K18. These changes ensure basal transcription of cell-cycle genes and are critical for their re-activation upon serum re-exposure. Our study reveals how direct manipulation of the epigenetic state of AEs by a general transcription factor regulates 3D genome folding and expression.

Scl represses cardiomyogenesis in prospective hemogenic endothelium and endocardium.

Endothelium in embryonic hematopoietic tissues generates hematopoietic stem/progenitor cells; however, it is unknown how its unique potential is specified. We show that transcription factor Scl/Tal1 is essential for both establishing the hematopoietic transcriptional program in hemogenic endothelium and preventing its misspecification to a cardiomyogenic fate. Scl(-/-) embryos activated a cardiac transcriptional program in yolk sac endothelium, leading to the emergence of CD31+Pdgfrα+ cardiogenic precursors that generated spontaneously beating cardiomyocytes. Ectopic cardiogenesis was also observed in Scl(-/-) hearts, where the disorganized endocardium precociously differentiated into cardiomyocytes. Induction of mosaic deletion of Scl in Scl(fl/fl)Rosa26Cre-ER(T2) embryos revealed a cell-intrinsic, temporal requirement for Scl to prevent cardiomyogenesis from endothelium. Scl(-/-) endothelium also upregulated the expression of Wnt antagonists, which promoted rapid cardiomyocyte differentiation of ectopic cardiogenic cells. These results reveal unexpected plasticity in embryonic endothelium such that loss of a single master regulator can induce ectopic cardiomyogenesis from endothelial cells.

A progesterone derivative linked to a stable phospholipid activates breast cancer cell response without leaving the cell membrane.

In hormone-responsive breast cancer cells, progesterone (P4) has been shown to act via its nuclear receptor (nPR), a ligand-activated transcription factor. A small fraction of progesterone receptor is palmitoylated and anchored to the cell membrane (mbPR) forming a complex with estrogen receptor alpha (ERα). Upon hormone exposure, either directly or via interaction with ERα, mbPR activates the SRC/RAS/ERK kinase pathway leading to phosphorylation of nPR by ERK. Kinase activation is essential for P4 gene regulation, as the ERK and MSK1 kinases are recruited by the nPR to its genomic binding sites and trigger chromatin remodeling. An interesting open question is whether activation of mbPR can result in gene regulation in the absence of ligand binding to intracellular progesterone receptor (iPR). This matter has been investigated in the past using P4 attached to serum albumin, but the attachment is leaky and albumin can be endocytosed and degraded, liberating P4. Here, we propose a more stringent approach to address this issue by ensuring attachment of P4 to the cell membrane via covalent binding to a stable phospholipid. This strategy identifies the actions of P4 independent from hormone binding to iPR. We found that a membrane-attached progestin can activate mbPR, the ERK signaling pathway leading to iPR phosphorylation, initial gene regulation and entry into the cell cycle, in the absence of detectable intracellular progestin.

New-onset atrial fibrillation and chronic coronary syndrome in the CLARIFY registry.

BACKGROUND AND AIMS: Data on new-onset atrial fibrillation (NOAF) in patients with chronic coronary syndromes (CCS) are scarce. This study aims to describe the incidence, predictors, and impact on cardiovascular (CV) outcomes of NOAF in CCS patients. METHODS: Data from the international (45 countries) CLARIFY registry (prospeCtive observational LongitudinAl RegIstry oF patients with stable coronary arterY disease) were used. Among 29 001 CCS outpatients without previously reported AF at baseline, patients with at least one episode of AF/flutter diagnosed during 5-year follow-up were compared with patients in sinus rhythm throughout the study. RESULTS: The incidence rate of NOAF was 1.12 [95% confidence interval (CI) 1.06-1.18] per 100 patient-years (cumulative incidence at 5 years: 5.0%). Independent predictors of NOAF were increasing age, increasing body mass index, low estimated glomerular filtration rate, Caucasian ethnicity, alcohol intake, and low left ventricular ejection fraction, while high triglycerides were associated with lower incidence. New-onset atrial fibrillation was associated with a substantial increase in the risk of adverse outcomes, with adjusted hazard ratios of 2.01 (95% CI 1.61-2.52) for the composite of CV death, non-fatal myocardial infarction, or non-fatal stroke, 2.61 (95% CI 2.04-3.34) for CV death, 1.64 (95% CI 1.07-2.50) for non-fatal myocardial infarction, 2.27 (95% CI 1.85-2.78) for all-cause death, 8.44 (95% CI 7.05-10.10) for hospitalization for heart failure, and 4.46 (95% CI 2.85-6.99) for major bleeding. CONCLUSIONS: Among CCS patients, NOAF is common and is strongly associated with worse outcomes. Whether more intensive preventive measures and more systematic screening for AF would improve prognosis in this population deserves further investigation.

Serum from COVID-19 patients promotes endothelial cell dysfunction through protease-activated receptor 2.

BACKGROUND: Endothelial dysfunction plays a central role in the pathophysiology of COVID-19 and is closely linked to the severity and mortality of the disease. The inflammatory response to SARS-CoV-2 infection can alter the capacity of the endothelium to regulate vascular tone, immune responses, and the balance between anti-thrombotic and pro-thrombotic properties. However, the specific endothelial pathways altered during COVID-19 still need to be fully understood. OBJECTIVE: In this study, we sought to identify molecular changes in endothelial cells induced by circulating factors characteristic of COVID-19. METHODS AND RESULTS: To this aim, we cultured endothelial cells with sera from patients with COVID-19 or non-COVID-19 pneumonia. Through transcriptomic analysis, we were able to identify a distinctive endothelial phenotype that is induced by sera from COVID-19 patients. We confirmed and expanded this observation in vitro by showing that COVID-19 serum alters functional properties of endothelial cells leading to increased apoptosis, loss of barrier integrity, and hypercoagulability. Furthermore, we demonstrated that these endothelial dysfunctions are mediated by protease-activated receptor 2 (PAR-2), as predicted by transcriptome network analysis validated by in vitro functional assays. CONCLUSION: Our findings provide the rationale for further studies to evaluate whether targeting PAR-2 may be a clinically effective strategy to counteract endothelial dysfunction in COVID-19.

C/EBPα mediates the growth inhibitory effect of progestins on breast cancer cells.

Steroid hormones are key gene regulators in breast cancer cells. While estrogens stimulate cell proliferation, progestins activate a single cell cycle followed by proliferation arrest. Here, we use biochemical and genome-wide approaches to show that progestins achieve this effect via a functional crosstalk with C/EBPα. Using ChIP-seq, we identify around 1,000 sites where C/EBPα binding precedes and helps binding of progesterone receptor (PR) in response to hormone. These regions exhibit epigenetic marks of active enhancers, and C/EBPα maintains an open chromatin conformation that facilitates loading of ligand-activated PR. Prior to hormone exposure, C/EBPα favors promoter-enhancer contacts that assure hormonal regulation of key genes involved in cell proliferation by facilitating binding of RAD21, YY1, and the Mediator complex. Knockdown of C/EBPα disrupts enhancer-promoter contacts and decreases the presence of these architectural proteins, highlighting its key role in 3D chromatin looping. Thus, C/EBPα fulfills a previously unknown function as a potential growth modulator in hormone-dependent breast cancer.

A set of accessible enhancers enables the initial response of breast cancer cells to physiological progestin concentrations.

Here, we report that in T47D breast cancer cells 50 pM progestin is sufficient to activate cell cycle entry and the progesterone gene expression program. At this concentration, equivalent to the progesterone blood levels found around the menopause, progesterone receptor (PR) binds only to 2800 genomic sites, which are accessible to ATAC cleavage prior to hormone exposure. These highly accessible sites (HAs) are surrounded by well-organized nucleosomes and exhibit breast enhancer features, including estrogen receptor alpha (ERα), higher FOXA1 and BRD4 (bromodomain containing 4) occupancy. Although HAs are enriched in RAD21 and CTCF, PR binding is the driving force for the most robust interactions with hormone-regulated genes. HAs show higher frequency of 3D contacts among themselves than with other PR binding sites, indicating colocalization in similar compartments. Gene regulation via HAs is independent of classical coregulators and ATP-activated remodelers, relying mainly on MAP kinase activation that enables PR nuclear engagement. HAs are also preferentially occupied by PR and ERα in breast cancer xenografts derived from MCF-7 cells as well as from patients, indicating their potential usefulness as targets for therapeutic intervention.

Cardiovascular prevention: sometimes dreams can come true.

Cardiovascular disease (CVD) is a chronic condition driven by the complex interaction of different risk factors including genetics, lifestyle, environment, etc. which, differently from other pathologies, can be prevented. Treatment of CVD has been inconceivably successful but now it seems that it has reached a plateau suggesting that prevention is the way forward. However, the COVID-19 pandemic has spotted all the limits of the actual health system regarding territorial and, particularly, of preventive medicine. To this end, recently, the SCORE2 risk prediction algorithms, a contemporary model to estimate 10 years risk of CVD in Europe and the new guidelines on prevention have been released. The present review article describes a dream: how prevention of CVD should be addressed in the future. New concepts and paradigms like early genetically personalized and imaging driven risk factors, cardiac risk cartography, measurements of the exposome, estimation of costs of a delayed outcome vs. healthy lifespan, are all addressed. We highlight the importance of technologies and the concept of being engaged in a 'healthy' and not just 'sick' system as it is today. The concept of 'clearing house' with a 'care health team' instead of a 'heart team' is described. Finally, we articulate the four points necessary for the dream to come true.

TFIIIC as a Potential Epigenetic Modulator of Histone Acetylation in Human Stem Cells.

Regulation of histone acetylation dictates patterns of gene expression and hence cell identity. Due to their clinical relevance in cancer biology, understanding how human embryonic stem cells (hESCs) regulate their genomic patterns of histone acetylation is critical, but it remains largely to be investigated. Here, we provide evidence that acetylation of histone H3 lysine-18 (H3K18ac) and lysine-27 (H3K27ac) is only partially established by p300 in stem cells, while it represents the main histone acetyltransferase (HAT) for these marks in somatic cells. Our analysis reveals that whereas p300 marginally associated with H3K18ac and H3K27ac in hESCs, it largely overlapped with these histone marks upon differentiation. Interestingly, we show that H3K18ac is found at "stemness" genes enriched in RNA polymerase III transcription factor C (TFIIIC) in hESCs, whilst lacking p300. Moreover, TFIIIC was also found in the vicinity of genes involved in neuronal biology, although devoid of H3K18ac. Our data suggest a more complex pattern of HATs responsible for histone acetylations in hESCs than previously considered, suggesting a putative role for H3K18ac and TFIIIC in regulating "stemness" genes as well as genes associated with neuronal differentiation of hESCs. The results break ground for possible new paradigms for genome acetylation in hESCs that could lead to new avenues for therapeutic intervention in cancer and developmental diseases.

International Observational Analysis of Evolution and Outcomes of Chronic Stable Angina: The Multinational CLARIFY Study.

BACKGROUND: Although angina is common in patients with stable coronary artery disease, limited data are available on its prevalence, natural evolution, and outcomes in the era of effective cardiovascular drugs and widespread use of coronary revascularization. METHODS: Using data from 32 691 patients with stable coronary artery disease from the prospective observational CLARIFY registry (Prospective Observational Longitudinal Registry of Patients with Stable Coronary Artery Disease), anginal status was mapped each year in patients without new coronary revascularization or new myocardial infarction. The use of medical interventions in the year preceding angina resolution was explored. The effect of 1-year changes in angina status on 5-year outcomes was analyzed using multivariable analysis. RESULTS: Among 7212 (22.1%) patients who reported angina at baseline, angina disappeared (without coronary revascularization) in 39.6% at 1 year, with further annual decreases. In patients without angina at baseline, 2.0% to 4.8% developed angina each year. During 5-year follow-up, angina was controlled in 7773 patients, in whom resolution of angina was obtained with increased use of antianginal treatment in 11.1%, with coronary revascularization in 4.5%, and without any changes in medication or revascularization in 84.4%. Compared to patients without angina at baseline and 1 year, persistence of angina and occurrence of angina at 1 year with conservative management were each independently associated with higher rates of cardiovascular death or myocardial infarction (adjusted hazard ratio, 1.32 [95% CI, 1.12-1.55] for persistence of angina; adjusted hazard ratio, 1.37 [95% CI, 1.11-1.70] for occurrence of angina) at 5 years. Patients whose angina had resolved at 1 year with conservative management were not at higher risk of cardiovascular death or myocardial infarction than those who never experienced angina (adjusted hazard ratio, 0.97 [95% CI, 0.82-1.15]). CONCLUSIONS: Angina affects almost one-quarter of patients with stable coronary artery disease but resolves without events or coronary revascularization in most patients. Resolution of angina within 1 year with conservative management predicted outcomes similar to lack of angina, whereas persistence or occurrence was associated with worse outcomes. Because most patients with angina are likely to experience resolution of symptoms, and because there is no demonstrated outcome benefit to routine revascularization, this study emphasizes the value of conservative management of stable coronary artery disease. Registration: URL: https://www.isrctn.com; Unique identifier: ISRCTN43070564.

Effects of Serelaxin in Patients with Acute Heart Failure.

BACKGROUND: Serelaxin is a recombinant form of human relaxin-2, a vasodilator hormone that contributes to cardiovascular and renal adaptations during pregnancy. Previous studies have suggested that treatment with serelaxin may result in relief of symptoms and in better outcomes in patients with acute heart failure. METHODS: In this multicenter, double-blind, placebo-controlled, event-driven trial, we enrolled patients who were hospitalized for acute heart failure and had dyspnea, vascular congestion on chest radiography, increased plasma concentrations of natriuretic peptides, mild-to-moderate renal insufficiency, and a systolic blood pressure of at least 125 mm Hg, and we randomly assigned them within 16 hours after presentation to receive either a 48-hour intravenous infusion of serelaxin (30 µg per kilogram of body weight per day) or placebo, in addition to standard care. The two primary end points were death from cardiovascular causes at 180 days and worsening heart failure at 5 days. RESULTS: A total of 6545 patients were included in the intention-to-treat analysis. At day 180, death from cardiovascular causes had occurred in 285 of the 3274 patients (8.7%) in the serelaxin group and in 290 of the 3271 patients (8.9%) in the placebo group (hazard ratio, 0.98; 95% confidence interval [CI], 0.83 to 1.15; P = 0.77). At day 5, worsening heart failure had occurred in 227 patients (6.9%) in the serelaxin group and in 252 (7.7%) in the placebo group (hazard ratio, 0.89; 95% CI, 0.75 to 1.07; P = 0.19). There were no significant differences between the groups in the incidence of death from any cause at 180 days, the incidence of death from cardiovascular causes or rehospitalization for heart failure or renal failure at 180 days, or the length of the index hospital stay. The incidence of adverse events was similar in the two groups. CONCLUSIONS: In this trial involving patients who were hospitalized for acute heart failure, an infusion of serelaxin did not result in a lower incidence of death from cardiovascular causes at 180 days or worsening heart failure at 5 days than placebo. (Funded by Novartis Pharma; RELAX-AHF-2 ClinicalTrials.gov number, NCT01870778.).

Hormone-control regions mediate steroid receptor-dependent genome organization.

In breast cancer cells, some topologically associating domains (TADs) behave as hormonal gene regulation units, within which gene transcription is coordinately regulated in response to steroid hormones. Here we further describe that responsive TADs contain 20- to 100-kb-long clusters of intermingled estrogen receptor (ESR1) and progesterone receptor (PGR) binding sites, hereafter called hormone-control regions (HCRs). In T47D cells, we identified more than 200 HCRs, which are frequently bound by unliganded ESR1 and PGR. These HCRs establish steady long-distance inter-TAD interactions between them and organize characteristic looping structures with promoters in their TADs even in the absence of hormones in ESR1+-PGR+ cells. This organization is dependent on the expression of the receptors and is further dynamically modulated in response to steroid hormones. HCRs function as platforms that integrate different signals, resulting in some cases in opposite transcriptional responses to estrogens or progestins. Altogether, these results suggest that steroid hormone receptors act not only as hormone-regulated sequence-specific transcription factors but also as local and global genome organizers.

Relationship between PCSK9 and endothelial function in patients with acute myocardial infarction.

BACKGROUND AND AIMS: While the role of PCSK9 in lipid metabolism is well established, its link with endothelial function is less clear. The aim of the present study is to evaluate the relationship between PCSK9 and endothelial dysfunction in the setting of acute myocardial infarction. METHODS AND RESULTS: To this purpose, we analyzed the serum of 74 patients with ST-elevation myocardial infarction (STEMI) at the time of admission and after 5 days. Endothelial dysfunction was evaluated as rate of apoptosis (AR) of human umbilical vein endothelial cells incubated with patients' serum. There was a good correlation between PCSK9 and the apoptosis rate values, both at baseline (r = 0.649) and 5-day (r = 0.648). In the 5 days after STEMI, PCSK9 increased significantly (242-327 ng/ml, p < 0.001), while AR did not (p = 0.491). Overall, 21 (28%) patients showed a reduction of PCSK9, and they had a significantly higher decrease of AR as compared to others (-13.87 vs 5.8%, p = 0.002). At the univariable analysis, the 5-day change of PCSK9 resulted to be the only variable associated with the 5-day change of the apoptosis rate (beta 0.217, 95%CI 0.091-0.344, p = 0.001). CONCLUSION: The variation of endothelial function and PCKS9 in the first days after an acute myocardial infarction are related. Further validation and research are necessary to confirm our findings. CLINICAL TRIAL: NCT02438085.

Cardiovascular prevention: sometimes dreams can come true.

Cardiovascular disease (CVD) is a chronic condition driven by the complex interaction of different risk factors including genetics, lifestyle, environment, etc. which, differently from other pathologies, can be prevented. Treatment of CVD has been inconceivably successful but now it seems that it has reached a plateau suggesting that prevention is the way forward. However, the COVID-19 pandemic has spotted all the limits of the actual health system regarding territorial and, particularly, of preventive medicine. To this end, recently, the SCORE2 risk prediction algorithms, a contemporary model to estimate 10-years risk of CVD in Europe and the new guidelines on prevention have been released. The present review article describes a dream: how prevention of CVD should be addressed in the future. New concepts and paradigms like early genetically personalized and imaging driven risk factors, cardiac risk cartography, measurements of the exposome, estimation of costs of a delayed outcome vs. healthy lifespan, are all addressed. We highlight the importance of technologies and the concept of being engaged in a 'healthy' and not just 'sick' system as it is today. The concept of 'clearing house' with a 'healthcare team' instead of a 'heart team' is described. Finally, we articulate the four points necessary for the dream to come true.

An RNA Polymerase III General Transcription Factor Engages in Cell Type-Specific Chromatin Looping.

Transcription factors (TFs) bind DNA in a sequence-specific manner and are generally cell type-specific factors and/or developmental master regulators. In contrast, general TFs (GTFs) are part of very large protein complexes and serve for RNA polymerases' recruitment to promoter sequences, generally in a cell type-independent manner. Whereas, several TFs have been proven to serve as anchors for the 3D genome organization, the role of GTFs in genome architecture have not been carefully explored. Here, we used ChIP-seq and Hi-C data to depict the role of TFIIIC, one of the RNA polymerase III GTFs, in 3D genome organization. We find that TFIIIC genome occupancy mainly occurs at specific regions, which largely correspond to Alu elements; other characteristic classes of repetitive elements (REs) such as MIR, FLAM-C and ALR/alpha are also found depending on the cell's developmental origin. The analysis also shows that TFIIIC-enriched regions are involved in cell type-specific DNA looping, which does not depend on colocalization with the master architectural protein CTCF. This work extends previous knowledge on the role of TFIIIC as a bona fide genome organizer whose action participates in cell type-dependent 3D genome looping via binding to REs.

Circulating Biomarkers of Endothelial Dysfunction and Inflammation in Predicting Clinical Outcomes in Diabetic Patients with Critical Limb Ischemia.

Critical limb ischemia (CLI) is a severe manifestation of peripheral artery disease characterized by ischemic pain, which is frequently associated with diabetes and non-healing lesions to inferior limbs. The clinical management of diabetic patients with CLI typically includes percutaneous transluminal angioplasty (PTA) to restore limb circulation and surgical treatment of diabetic foot ulcers (DFU). However, even after successful treatment, CLI patients are prone to post-procedure complications, which may lead to unplanned revascularization or foot surgery. Unfortunately, the factors predicting adverse events in treated CLI patients are only partially known. This study aimed to identify potential biomarkers that predict the disease course in diabetic patients with CLI. For this purpose, we measured the circulating levels of a panel of 23 molecules related to inflammation, endothelial dysfunction, platelet activation, and thrombophilia in 92 patients with CLI and DFU requiring PTA and foot surgery. We investigated whether these putative biomarkers were associated with the following clinical endpoints: (1) healing of the treated DFUs; (2) need for new revascularization of the limb; (3) appearance of new lesions or relapses after successful healing. We found that sICAM-1 and endothelin-1 are inversely associated with DFU healing and that PAI-1 and endothelin-1 are associated with the need for new revascularization. Moreover, we found that the levels of thrombomodulin and sCD40L are associated with new lesions or recurrence, and we show that the levels of these biomarkers could be used in a decision tree to assign patients to clusters with different risks of developing new lesions or recurrences.

Efficacy and safety of trimetazidine after percutaneous coronary intervention (ATPCI): a randomised, double-blind, placebo-controlled trial.

BACKGROUND: Angina might persist or reoccur despite successful revascularisation with percutaneous coronary intervention (PCI) and antianginal therapy. Additionally, PCI in stable patients has not been shown to improve survival compared with optimal medical therapy. Trimetazidine is an antianginal agent that improves energy metabolism of the ischaemic myocardium and might improve outcomes and symptoms of patients who recently had a PCI. In this study, we aimed to assess the long-term potential benefits and safety of trimetazidine added to standard evidence-based medical treatment in patients who had a recent successful PCI. METHODS: We did a randomised, double-blind, placebo-controlled, event-driven trial of trimetazidine added to standard background therapy in patients who had undergone successful PCI at 365 centres in 27 countries across Europe, South America, Asia, and north Africa. Eligible patients were aged 21-85 years and had had either elective PCI for stable angina or urgent PCI for unstable angina or non-ST segment elevation myocardial infarction less than 30 days before randomisation. Patients were randomly assigned by an interactive web response system to oral trimetazidine 35 mg modified-release twice daily or matching placebo. Participants, study investigators, and all study staff were masked to treatment allocation. The primary efficacy endpoint was a composite of cardiac death; hospital admission for a cardiac event; recurrence or persistence of angina requiring an addition, switch, or increase of the dose of at least one antianginal drug; or recurrence or persistence of angina requiring a coronary angiography. Efficacy analyses were done according to the intention-to-treat principle. Safety was assessed in all patients who had at least one dose of study drug. This study is registered with the EU Clinical Trials Register (EudraCT 2010-022134-89). FINDINGS: From Sept 17, 2014, to June 15, 2016, 6007 patients were enrolled and randomly assigned to receive either trimetazidine (n=2998) or placebo (n=3009). After a median follow-up of 47·5 months (IQR 42·3-53·3), incidence of primary endpoint events was not significantly different between the trimetazidine group (700 [23·3%] patients) and the placebo group (714 [23·7%]; hazard ratio 0·98 [95% CI 0·88-1·09], p=0·73). When analysed individually, there were no significant differences in the incidence of the components of the primary endpoint between the treatment groups. Similar results were obtained when patients were categorised according to whether they had an elective or urgent PCI. 1219 (40·9%) of 2983 patients in the trimetazidine group and 1230 (41·1%) of 2990 patients in the placebo group had serious treatment-emergent adverse events. Frequencies of adverse events of interest were similar between the groups. INTERPRETATION: Our results show that the routine use of oral trimetazidine 35 mg twice daily over several years in patients receiving optimal medical therapy, after successful PCI, does not influence the recurrence of angina or the outcome; these findings should be taken into account when considering the place of trimetazidine in clinical practice. However, the long-term prescription of this treatment does not appear to be associated with any statistically significant safety concerns in the population studied. FUNDING: Servier.

Markers of endothelial and epithelial pulmonary injury in mechanically ventilated COVID-19 ICU patients.

BACKGROUND: Biomarkers can be used to detect the presence of endothelial and/or alveolar epithelial injuries in case of ARDS. Angiopoietin-2 (Ang-2), soluble intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion protein-1 (VCAM-1), P-selectin and E-selectin are biomarkers of endothelial injury, whereas the receptor for advanced glycation end-products (RAGE) reflects alveolar epithelial injury. The aims of this study were to evaluate whether the plasma concentration of the above-mentioned biomarkers was different 1) in survivors and non-survivors of COVID-19-related ARDS and 2) in COVID-19-related and classical ARDS. METHODS: This prospective study was performed in two COVID-19-dedicated Intensive Care Units (ICU) and one non-COVID-19 ICU at Ferrara University Hospital. A cohort of 31 mechanically ventilated patients with COVID-19 ARDS and a cohort of 11 patients with classical ARDS were enrolled. Ang-2, ICAM-1, VCAM-1, P-selectin, E-selectin and RAGE were determined with a bead-based multiplex immunoassay at three time points: inclusion in the study (T1), after 7 ± 2 days (T2) and 14 ± 2 days (T3). The primary outcome was to evaluate the plasma trend of the biomarker levels in survivors and non-survivors. The secondary outcome was to evaluate the differences in respiratory mechanics variables and gas exchanges between survivors and non-survivors. Furthermore, we compared the plasma levels of the biomarkers at T1 in patients with COVID-19-related ARDS and classical ARDS. RESULTS: In COVID-19-related ARDS, the plasma levels of Ang-2 and ICAM-1 at T1 were statistically higher in non-survivors than survivors, (p = 0.04 and p = 0.03, respectively), whereas those of P-selectin, E-selectin and RAGE did not differ. Ang-2 and ICAM-1 at T1 were predictors of mortality (AUROC 0.650 and 0.717, respectively). At T1, RAGE and P-selectin levels were higher in classical ARDS than in COVID-19-related ARDS. Ang-2, ICAM-1 and E-selectin were lower in classical ARDS than in COVID-19-related ARDS (all p < 0.001). CONCLUSIONS: COVID-19 ARDS is characterized by an early pulmonary endothelial injury, as detected by Ang-2 and ICAM-1. COVID-19 ARDS and classical ARDS exhibited a different expression of biomarkers, suggesting different pathological pathways. Trial registration NCT04343053 , Date of registration: April 13, 2020.

Histone acetylation regulates intracellular pH.

Differences in global levels of histone acetylation occur in normal and cancer cells, although the reason why cells regulate these levels has been unclear. Here we demonstrate a role for histone acetylation in regulating intracellular pH (pH(i)). As pH(i) decreases, histones are globally deacetylated by histone deacetylases (HDACs), and the released acetate anions are coexported with protons out of the cell by monocarboxylate transporters (MCTs), preventing further reductions in pH(i). Conversely, global histone acetylation increases as pH(i) rises, such as when resting cells are induced to proliferate. Inhibition of HDACs or MCTs decreases acetate export and lowers pH(i), particularly compromising pH(i) maintenance in acidic environments. Global deacetylation at low pH is reflected at a genomic level by decreased abundance and extensive redistribution of acetylation throughout the genome. Thus, acetylation of chromatin functions as a rheostat to regulate pH(i) with important implications for mechanism of action and therapeutic use of HDAC inhibitors.