RUNX1: an emerging therapeutic target for cardiovascular disease

Runt-related transcription factor-1 (RUNX1), also known as acute myeloid leukaemia 1 protein (AML1), is a member of the core-binding factor family of transcription factors which modulate cell proliferation, differentiation, and survival in multiple systems. It is a master-regulator transcription factor, which has been implicated in diverse signalling pathways and cellular mechanisms during normal development and disease. RUNX1 is best characterized for its indispensable role for definitive haematopoiesis and its involvement in haematological malignancies. However, more recently RUNX1 has been identified as a key regulator of adverse cardiac remodelling following myocardial infarction. This review discusses the role RUNX1 plays in the heart and highlights its therapeutic potential as a target to limit the progression of adverse cardiac remodelling and heart failure

Machine learning for classification of hypertension subtypes using multi-omics: a multi-centre, retrospective, data-driven study

Background: Arterial hypertension is a major cardiovascular risk factor. Identification of secondary hypertension in its various forms is key to preventing and targeting treatment of cardiovascular complications. Simplified diagnostic tests are urgently required to distinguish primary and secondary hypertension to address the current underdiagnosis of the latter. Methods: This study uses Machine Learning (ML) to classify subtypes of endocrine hypertension (EHT) in a large cohort of hypertensive patients using multidimensional omics analysis of plasma and urine samples. We measured 409 multi-omics (MOmics) features including plasma miRNAs (PmiRNA: 173), plasma catechol O-methylated metabolites (PMetas: 4), plasma steroids (PSteroids: 16), urinary steroid metabolites (USteroids: 27), and plasma small metabolites (PSmallMB: 189) in primary hypertension (PHT) patients, EHT patients with either primary aldosteronism (PA), pheochromocytoma/functional paraganglioma (PPGL) or Cushing syndrome (CS) and normotensive volunteers (NV). Biomarker discovery involved selection of disease combination, outlier handling, feature reduction, 8 ML classifiers, class balancing and consideration of different age- and sex-based scenarios. Classifications were evaluated using balanced accuracy, sensitivity, specificity, AUC, F1, and Kappa score. Findings: Complete clinical and biological datasets were generated from 307 subjects (PA=113, PPGL=88, CS=41 and PHT=112). The random forest classifier provided ∼92% balanced accuracy (∼11% improvement on the best mono-omics classifier), with 96% specificity and 0.95 AUC to distinguish one of the four conditions in multi-class ALL-ALL comparisons (PPGL vs PA vs CS vs PHT) on an unseen test set, using 57 MOmics features. For discrimination of EHT (PA + PPGL + CS) vs PHT, the simple logistic classifier achieved 0.96 AUC with 90% sensitivity, and ∼86% specificity, using 37 MOmics features. One PmiRNA (hsa-miR-15a-5p) and two PSmallMB (C9 and PC ae C38:1) features were found to be most discriminating for all disease combinations. Overall, the MOmics-based classifiers were able to provide better classification performance in comparison to mono-omics classifiers. Interpretation: We have developed a ML pipeline to distinguish different EHT subtypes from PHT using multi-omics data. This innovative approach to stratification is an advancement towards the development of a diagnostic tool for EHT patients, significantly increasing testing throughput and accelerating administration of appropriate treatment. Funding: European Union's Horizon 2020 Research and Innovation Programme under Grant Agreement No. 633983, Clinical Research Priority Program of the University of Zurich for the CRPP HYRENE (to Z.E. and F.B.), and Deutsche Forschungsgemeinschaft (CRC/Transregio 205/1)

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Characterisation of the role of RUNX1 in the context of myocardial ischaemia-reperfusion injury

Coronary artery disease is the leading cause of death and disability worldwide and is typically caused by atherosclerotic narrowing of the coronary arteries, which impairs blood flow (ischaemia) to heart. In severe cases atherosclerotic plaques destabilise and rupture, resulting in complete occlusion of the coronary artery and progressive ischaemic necrosis (myocardial infarction, MI). The optimal treatment is to restore the patency of the coronary artery using primary percutaneous coronary intervention (PPCI) to reperfuse the ischaemic myocardium and limit necrosis. However, paradoxically, the full efficacy of PPCI is limited by additional injury initiated by reperfusion that causes cell death and contributes to contractile dysfunction. The collective injury caused by ischaemia and reperfusion, termed ischaemia-reperfusion (I/R) injury, leaves patients vulnerable to adverse cardiac remodelling and the development of heart failure, a hugely debilitating condition with high mortality rates. As such, there is a pressing, unmet need for novel therapeutic strategies that can be used as an adjunctive to reperfusion therapy to limit contractile dysfunction and adverse remodelling post-MI and prevent heart failure development. Previous work has highlighted RUNX1 as a promising therapeutic target for the management of I/R injury and adverse cardiac remodelling. RUNX1 belongs to the RUNX family of transcription factors and is upregulated in border zone cardiomyocytes following MI. Using mice with a cardiomyocyte specific deficiency in Runx1 (Runx1∆/∆ mice) it was previously shown that RUNX1 is detrimental to contractility and contributes to adverse cardiac remodelling in experimental models of MI and I/R injury. It was subsequently demonstrated that increased contractility of Runx1∆/∆ hearts at 2 weeks post-MI was explained by improved cardiomyocyte calcium handling, caused by a decrease in protein phosphatase 1 (PP1) expression, increased phosphorylation of phospholamban (PLB) and the relief of sarcoplasmic reticulum calcium ATPase (SERCA) inhibition. This thesis aimed to further investigate the role of RUNX1 in the context of myocardial I/R injury. Specifically, it aimed to study potential triggers for Runx1 mRNA that may arise in the infarct and border zone including I/R injury and stretch. It also aimed to explore whether Runx1 deficiency offers acute (<24 h) protection against contractile dysfunction in response to I/R injury and whether this was linked to altered calcium handling protein expression. To fulfil these aims, a mouse Langendorff model was established to allow evaluation of the acute responses of isolated hearts to I/R injury and myocardial stretch in the ex vivo setting. Two separate models of I/R injury were characterised: i) no-flow I/R injury where perfusion was completely halted during ischaemia and ii) low-flow I/R injury where residual perfusion was provided during ischaemia to mimic perfusion via collateral vessels. Isolated hearts demonstrated a robust and consistent response to these protocols that paralleled findings reported by others in the field. In addition, the results demonstrated that lengthening the duration of no-flow ischaemia increased the final infarct size, corroborating the work of others and further validating the I/R protocol used. Using the Langendorff set up, it was shown that perfusion was sufficient to induce rapid Runx1 mRNA upregulation, indicating that changes in Runx1 mRNA expression can occur within hours in the isolated heart, independently of neuroendocrine or systemic inflammatory interactions. No-flow I/R injury but not low-flow I/R injury, decreased Runx1 mRNA expression relative to time-matched continuously perfused control hearts. At the same time, the mRNA expression of Runx2 and Runx3 was also measured. Runx2 expression did not change in any of the conditions tested; however, Runx3 mRNA expression was decreased by perfusion, and this downregulation could be offset by no-flow I/R injury but not low-flow I/R injury. Using an intraventricular balloon to stretch the left ventricle of isolated hearts, we investigated whether stretch stimuli affected Runx1 mRNA. Interestingly, intermittent stretch (cycles of balloon inflation and deflation) but not sustained stretch was able to offset the upregulation of Runx1 mRNA by perfusion, indicating that stretch can regulate Runx1 expression when present as a dynamic stimulus. Using the Langendorff model of no-flow I/R injury, it was next demonstrated that Runx1∆/∆ hearts had improved post-ischaemic contractile recovery compared to control hearts, indicating, for the first time, that the protective effects of Runx1 deficiency manifest within an hour of I/R injury and occur in isolation from systemic stimuli. There was no significant difference between PLB, phosphorylated PLB at the serine 16 residue or PP1 expression between Runx1∆/∆ and control hearts either at the beginning or end of the reperfusion period. Taken together, this suggests that the effect of Runx1 deficiency on contractility post-Langendorff I/R injury occurs via a distinct mechanism from that at 2 weeks post-MI. In the final part of this project, the role of Runx1 in hypoxic responses in neonatal rat cardiomyocytes (NRCMs) was investigated. To explore whether Runx mRNAs are upregulated alongside a hypoxic gene profile, NRCMs were treated with hypoxic mimetics. Dimethyloxalyglycine (DMOG) did not affect the expression of Runx mRNAs, whereas cobalt chloride (CoCl2) and deferoxamine (DFO) selectively upregulated Runx1 and Runx3 mRNA, respectively. Further work is necessary to understand the different effects of these mimetics on the Runx family. In non-cardiac tissue, physical and functional interactions exist between hypoxia inducible factor 1α (HIF-1α) and RUNX1. As HIF1-α directs the transcription of a large set of genes (including VEGF and GLUT1) as part of the cellular response to ischaemia, we explored whether RUNX1 expression levels modulate expression of HIF-1α target transcripts in NRCMs. However, RUNX1 overexpression did not affect the mRNA expression of HIF-1α target genes VEGF or GLUT1 under baseline conditions or in the presence of CoCl2. Overall, the work in this thesis has confirmed that the beneficial effects of Runx1 deficiency previously found in vivo extend to the ex vivo setting and manifest acutely during reperfusion. It was demonstrated that a range of stimuli can alter Runx1 mRNA in the isolated heart, including intermittent stretch, which has not previously been linked to Runx1 mRNA regulation in the heart. Developing these findings further will be key to understanding myocardial RUNX1 function and the translational potential of targeting RUNX1 as a therapeutic strategy in the future

CNS border-associated macrophages in the homeostatic and ischaemic brain

CNS border-associated macrophages (BAMs) are a small population of specialised macrophages localised in the choroid plexus, meningeal and perivascular spaces. Until recently, the function of this elusive cell type was poorly understood and largely overlooked, especially in comparison to microglia, the primary brain resident immune cell. However, the recent single cell immunophenotyping or transcriptomic analysis of BAM subsets in the homeostatic brain, coupled with the rapid emergence of new studies exploring BAM functions in various cerebral pathologies, including Alzheimer's disease, hypertension-induced neurovascular and cognitive dysfunction, and ischaemic stroke, has unveiled previously unrecognised heterogeneity and spatial-temporal complexity in BAM populations as well as their contributions to brain homeostasis and disease. In this review, we discuss the implications of this new-found knowledge on our current understanding of BAM function in ischaemic stroke. We first provide a comprehensive overview and discussion of the cell-surface expression profiles, transcriptional signatures and potential functional phenotypes of homeostatic BAM subsets described in recent studies. Evidence for their putative physiological roles is examined, including their involvement in immunological surveillance, waste clearance, and vascular permeability. We discuss the evidence supporting the accumulation and genetic transformation of BAMs in response to ischaemia and appraise the experimental evidence that BAM function might be deleterious in the acute phase of stroke, while considering the mechanisms by which BAMs may influence stroke outcomes in the longer term. Finally, we review the therapeutic potential of immunomodulatory strategies as an approach to stroke management, highlighting current challenges in the field and key issues relating to BAMs, and how BAMs could be harnessed experimentally to support future translational research

Regulation of connexin 43 by interleukin 1β in adult rat cardiac fibroblasts and effects in an adult rat cardiac myocyte: fibroblast co-culture model

Connexin 43 expression (Cx43) is increased in cardiac fibroblasts (CFs) following myocardial infarction. Here, potential mediators responsible for increasing Cx43 expression and effects of differential CF phenotype on cardiac myocyte (CM) function were investigated. Stimulating adult rat CFs with proinflammatory mediators revealed that interleukin 1β (IL-1β) significantly enhanced Cx43 levels through the IL-1β pathway. Additionally, IL-1β reduced mRNA levels of the myofibroblast (MF) markers: (i) connective tissue growth factor (CTGF) and (ii) α smooth muscle actin (αSMA), compared to control CFs. A co-culture adult rat CM:CF model was utilised to examine cell-to-cell interactions. Transfer of calcein from CMs to underlying CFs suggested functional gap junction formation. Functional analysis revealed contraction duration (CD) of CMs was shortened in co-culture with CFs, while treatment of CFs with IL-1β reduced this mechanical effect of co-culture. No effect on action potential rise time or duration of CMs cultured with control or IL-1β-treated CFs was observed. These data demonstrate that stimulating CFs with IL-1β increases Cx43 and reduces MF marker expression, suggesting altered cell phenotype. These changes may underlie the reduced mechanical effects of IL-1β treated CFs on CD of co-cultured CMs and therefore have an implication for our understanding of heterocellular interactions in cardiac disease