A novel, small-volume subcutaneous furosemide formulation delivered by an abdominal patch infusor device in patients with heart failure: results of two phase I studies.

AIMS: Subcutaneous (SC) furosemide has potential advantages over intravenous (IV) furosemide by enabling self-administration or administration by a lay caregiver, such as facilitating early discharge, preventing hospitalizations, and in palliative care. A high-concentration, pH-neutral furosemide formulation has been developed for SC administration via a small patch infusor pump. We aimed to compare the bioavailability, pharmacokinetic (PK), and pharmacodynamic (PD) profiles of a new SC furosemide formulation with conventional IV furosemide and describe the first use of a bespoke mini-pump to administer this formulation. METHODS AND RESULTS: A novel pH-neutral formulation of SC furosemide containing 80 mg furosemide in ∼2.7 mL (infused over 5 h) was investigated. The first study was a PK/PD study of SC furosemide compared with 80 mg IV furosemide administered as a bolus in ambulatory patients with heart failure (HF). The primary outcome was absolute bioavailability of SC compared with IV furosemide. The second study investigated the same SC furosemide preparation delivered by a patch infusor in patients hospitalized with HF. Primary outcome measures were treatment-emergent adverse events, infusion site pain, device performance, and PK measurements.The absolute bioavailability of SC furosemide in comparison to IV furosemide was 112%, resulting in equivalent diuresis and natriuresis. When SC furosemide was administered via the patch pump, there were no treatment-emergent adverse events and 95% of participants reported no/minor discomfort at the infusion site. CONCLUSION: The novel preparation of SC furosemide had similar bioavailability to IV furosemide. Administration via a patch pump was feasible and well tolerated.

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).

Implementation of cancer screening in rural primary care practices after joining an accountable care organisation: a multiple case study.

OBJECTIVE: To describe common strategies and practice-specific barriers, adaptations and determinants of cancer screening implementation in eight rural primary care practices in the Midwestern United States after joining an accountable care organisation (ACO). DESIGN: This study used a multiple case study design. Purposive sampling was used to identify a diverse group of practices within the ACO. Data were collected from focus group interviews and workflow mapping. The Consolidated Framework for Implementation Research (CFIR) was used to guide data collection and analysis. Data were cross-analysed by clinic and CFIR domains to identify common themes and practice-specific determinants of cancer screening implementation. SETTING: The study included eight rural primary care practices, defined as Rural-Urban Continuum Codes 5-9, in one ACO in the Midwestern United States. PARTICIPANTS: Providers, staff and administrators who worked in the primary care practices participated in focus groups. 28 individuals participated including 10 physicians; one doctor of osteopathic medicine; three advanced practice registered nurses; eight registered nurses, quality assurance and licensed practical nurses; one medical assistant; one care coordination manager; and four administrators. RESULTS: With integration into the ACO, practices adopted four new strategies to support cancer screening: care gap lists, huddle sheets, screening via annual wellness visits and information spread. Cross-case analysis revealed that all practices used both visit-based and population-based cancer screening strategies, although workflows varied widely across practices. Each of the four strategies was adapted for fit to the local context of the practice. Participants shared that joining the ACO provided a strong external incentive for increasing cancer screening rates. Two predominant determinants of cancer screening success at the clinic level were use of the electronic health record (EHR) and fully engaging nurses in the screening process. CONCLUSIONS: Joining an ACO can be a positive driver for increasing cancer screening practices in rural primary care practices. Characteristics of the practice can impact the success of ACO-related cancer screening efforts; engaging nurses to the fullest extent of their education and training and integrating cancer screening into the EHR can optimise the cancer screening workflow.

Circulating microRNAs as Diagnostic Markers in Primary Aldosteronism.

Primary aldosteronism (PA) is a common and highly treatable condition, usually resulting from adrenocortical tumorous growth or hyperplasia. PA is currently underdiagnosed owing to its complex and protracted diagnostic procedures. A simplified biomarker-based test would be highly valuable in reducing cardiovascular morbidity and mortality. Circulating microRNAs are emerging as potential biomarkers for a number of conditions due to their stability and accessibility. PA is known to alter microRNA expression in adrenocortical tissue; if these changes or their effects are mirrored in the circulating miRNA profile, then this could be exploited by a diagnostic test. However, the reproducibility of studies to identify biomarker-circulating microRNAs has proved difficult for other conditions due to a series of technical challenges. Therefore, any studies seeking to definitively identify circulating microRNA biomarkers of PA must address this in their design. To this end, we are currently conducting the circulating microRNA arm of the ongoing ENS@T-HT study. In this review article, we present evidence to support the utility of circulating microRNAs as PA biomarkers, describe the practical challenges to this approach and, using ENS@T-HT as an example, discuss how these might be overcome.

TRAIL-Expressing Monocyte/Macrophages Are Critical for Reducing Inflammation and Atherosclerosis.

Circulating tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) levels are reduced in patients with cardiovascular disease, and TRAIL gene deletion in mice exacerbates atherosclerosis and inflammation. How TRAIL protects against atherosclerosis and why levels are reduced in disease is unknown. Here, multiple strategies were used to identify the protective source of TRAIL and its mechanism(s) of action. Samples from patients with coronary artery disease and bone-marrow transplantation experiments in mice lacking TRAIL revealed monocytes/macrophages as the main protective source. Accordingly, deletion of TRAIL caused a more inflammatory macrophage with reduced migration, displaying impaired reverse cholesterol efflux and efferocytosis. Furthermore, interleukin (IL)-18, commonly increased in plasma of patients with cardiovascular disease, negatively regulated TRAIL transcription and gene expression, revealing an IL-18-TRAIL axis. These findings demonstrate that TRAIL is protective of atherosclerosis by modulating monocyte/macrophage phenotype and function. Manipulating TRAIL levels in these cells highlights a different therapeutic avenue in the treatment of cardiovascular disease.

VEGFR2 is activated by high-density lipoproteins and plays a key role in the proangiogenic action of HDL in ischemia.

High-density lipoproteins augment hypoxia-induced angiogenesis by inducing the key angiogenic vascular endothelial growth factor A (VEGFA) and total protein levels of its receptor 2 (VEGFR2). The activation/phosphorylation of VEGFR2 is critical for mediating downstream, angiogenic signaling events. This study aimed to determine whether reconstituted high-density lipoprotein (rHDL) activates VEGFR2 phosphorylation and the downstream signaling events and the importance of VEGFR2 in the proangiogenic effects of rHDL in hypoxia. In vitro, rHDL increased VEGFR2 activation and enhanced phosphorylation of downstream, angiogenic signaling proteins ERK1/2 and p38 MAPK in hypoxia. Incubation with a VEGFR2-neutralizing antibody attenuated rHDL-induced phosphorylation of VEGFR2, ERK1/2, p38 MAPK, and tubule formation. In a murine model of ischemia-driven neovascularization, rHDL infusions enhanced blood perfusion and augmented capillary and arteriolar density. Infusion of a VEGFR2-neutralizing antibody ablated those proangiogenic effects of rHDL. Circulating Sca1+/CXCR4+ angiogenic progenitor cell levels, important for neovascularization in response to ischemia, were higher in rHDL-infused mice 3 d after ischemic induction, but that did not occur in mice that also received the VEGFR2-neutralizing antibody. In summary, VEGFR2 has a key role in the proangiogenic effects of rHDL in hypoxia/ischemia. These findings have therapeutic implications for angiogenic diseases associated with an impaired response to tissue ischemia.-Cannizzo, C. M., Adonopulos, A. A., Solly, E. L., Ridiandries, A., Vanags, L. Z., Mulangala, J., Yuen, S. C. G., Tsatralis, T., Henriquez, R., Robertson, S., Nicholls, S. J., Di Bartolo, B. A., Ng, M. K. C., Lam, Y. T., Bursill, C. A., Tan, J. T. M. VEGFR2 is activated by high-density lipoproteins and plays a key role in the proangiogenic action of HDL in ischemia.

Regulation of Corticosteroidogenic Genes by MicroRNAs.

The loss of normal regulation of corticosteroid secretion is important in the development of cardiovascular disease. We previously showed that microRNAs regulate the terminal stages of corticosteroid biosynthesis. Here, we assess microRNA regulation across the whole corticosteroid pathway. Knockdown of microRNA using Dicer1 siRNA in H295R adrenocortical cells increased levels of CYP11A1, CYP21A1, and CYP17A1 mRNA and the secretion of cortisol, corticosterone, 11-deoxycorticosterone, 18-hydroxycorticosterone, and aldosterone. Bioinformatic analysis of genes involved in corticosteroid biosynthesis or metabolism identified many putative microRNA-binding sites, and some were selected for further study. Manipulation of individual microRNA levels demonstrated a direct effect of miR-125a-5p and miR-125b-5p on CYP11B2 and of miR-320a-3p levels on CYP11A1 and CYP17A1 mRNA. Finally, comparison of microRNA expression profiles from human aldosterone-producing adenoma and normal adrenal tissue showed levels of various microRNAs, including miR-125a-5p to be significantly different. This study demonstrates that corticosteroidogenesis is regulated at multiple points by several microRNAs and that certain of these microRNAs are differentially expressed in tumorous adrenal tissue, which may contribute to dysregulation of corticosteroid secretion. These findings provide new insights into the regulation of corticosteroid production and have implications for understanding the pathology of disease states where abnormal hormone secretion is a feature.

Neutrophil-derived microparticles are released into the coronary circulation following percutaneous coronary intervention in acute coronary syndrome patients.

To evaluate (i) local coronary and systemic levels of microparticles (MP) in acute coronary syndrome (ACS) and stable angina pectoris (SAP) patients and (ii) their release after plaque disruption with percutaneous coronary intervention (PCI). MP are small vesicles originating from plasma membranes of cells after activation or apoptosis and are implicated in the pathogenesis of atherosclerosis. Neutrophils play a role in plaque destabilization and shed neutrophil-derived MP that have the potential to drive significant proinflammatory and thrombotic downstream effects. Eight ACS and eight SAP patients were included. Coronary sinus (CS) samples pre-intervention (CS1), 45 s following balloon angioplasty (CS2) and at 45 s intervals following stent deployment (CS3, CS4 and CS5), together with peripheral vein samples, pre- and post-PCI were analysed for neutrophil-derived (CD66b+), endothelial-derived (CD144+), platelet-derived (CD41a+), monocyte-derived (CD14+) and apoptotic (Annexin V+) MP. ELISA for interleukin (IL)-6, myeloperoxidase (MPO) and P-selectin was also performed. CD66b+ MP levels were similar in both groups pre-intervention. Post-PCI, CS levels rose significantly in ACS but not SAP patients (ACS area under the curve (AUC): 549 ± 83, SAP AUC: 24 ± 29, P<0.01). CS CD41a+, CD144+, CD14+ and Annexin V+ MP levels did not differ between groups. Acute neutrophil-derived MP release post-PCI occurs in ACS compared with stable patients, likely to be reflective of plaque MP content in vulnerable lesions.

Reconstituted high-density lipoproteins promote wound repair and blood flow recovery in response to ischemia in aged mice.

BACKGROUND: The average population age is increasing and the incidence of age-related vascular complications is rising in parallel. Impaired wound healing and disordered ischemia-mediated angiogenesis are key contributors to age-impaired vascular complications that can lead to amputation. High-density lipoproteins (HDL) have vasculo-protective properties and augment ischemia-driven angiogenesis in young animals. We aimed to determine the effect of reconstituted HDL (rHDL) on aged mice in a murine wound healing model and the hindlimb ischemia (HLI) model. METHODS: Murine wound healing model-24-month-old aged mice received topical application of rHDL (50 µg/wound/day) or PBS (vehicle control) for 10 days following wounding. Murine HLI model-Femoral artery ligation was performed on 24-month-old mice. Mice received rHDL (40 mg/kg) or PBS, intravenously, on alternate days, 1 week pre-surgery and up to 21 days post ligation. For both models, blood flow perfusion was determined using laser Doppler perfusion imaging. Mice were sacrificed at 10 (wound healing) or 21 (HLI) days post-surgery and tissues were collected for histological and gene analyses. RESULTS: Daily topical application of rHDL increased the rate of wound closure by Day 7 post-wounding (25 %, p < 0.05). Wound blood perfusion, a marker of angiogenesis, was elevated in rHDL treated wounds (Days 4-10 by 22-25 %, p < 0.05). In addition, rHDL increased wound capillary density by 52.6 %. In the HLI model, rHDL infusions augmented blood flow recovery in ischemic limbs (Day 18 by 50 % and Day 21 by 88 %, p < 0.05) and prevented tissue necrosis and toe loss. Assessment of capillary density in ischemic hindlimb sections found a 90 % increase in rHDL infused animals. In vitro studies in fibroblasts isolated from aged mice found that incubation with rHDL was able to significantly increase the key pro-angiogenic mediator vascular endothelial growth factor (VEGF) protein (25 %, p < 0.05). CONCLUSION: rHDL can promote wound healing and wound angiogenesis, and blood flow recovery in response to ischemia in aged mice. Mechanistically, this is likely to be via an increase in VEGF. This highlights a potential role for HDL in the therapeutic modulation of age-impaired vascular complications.

Retargeting FX-binding-ablated HAdV-5 to vascular cells by inclusion of the RGD-4C peptide in hexon hypervariable region 7 and the HI loop.

Recent studies have generated interest in the function of human adenovirus serotype 5 (HAdV-5) hexon: factor X (FX) binding and subsequent hepatocyte transduction and interaction with the immune system. Here, we retargeted adenovirus serotype 5 vectors, ablated for FX interaction, by replacing amino acids in hexon HVR7 with RGD-4C or inserting the peptide into the fibre HI loop. These genetic modifications in the capsid were compatible with virus assembly, and could efficiently retarget transduction of the vector via the αvß3/5 integrin-mediated pathway, but did not alter immune recognition by pre-existing human neutralizing anti-HAdV-5 antibodies or by natural antibodies in mouse serum. Thus, FX-binding-ablated HAdV-5 can be retargeted but remain sensitive to immune-mediated attack. These findings further refine HAdV-5-based vectors for human gene therapy and inform future vector development.

Colchicine Acutely Suppresses Local Cardiac Production of Inflammatory Cytokines in Patients With an Acute Coronary Syndrome.

BACKGROUND: Interleukin (IL)-1ß, IL-18, and downstream IL-6 are key inflammatory cytokines in the pathogenesis of coronary artery disease. Colchicine is believed to block the NLRP3 inflammasome, a cytosolic complex responsible for the production of IL-1ß and IL-18. In vivo effects of colchicine on cardiac cytokine release have not been previously studied. This study aimed to (1) assess the local cardiac production of inflammatory cytokines in patients with acute coronary syndromes (ACS), stable coronary artery disease and in controls; and (2) determine whether acute administration of colchicine inhibits their production. METHODS AND RESULTS: Forty ACS patients, 33 with stable coronary artery disease, and 10 controls, were included. ACS and stable coronary artery disease patients were randomized to oral colchicine treatment (1 mg followed by 0.5 mg 1 hour later) or no colchicine, 6 to 24 hours prior to cardiac catheterization. Blood samples from the coronary sinus, aortic root (arterial), and lower right atrium (venous) were collected and tested for IL-1ß, IL-18, and IL-6 using ELISA. In ACS patients, coronary sinus levels of IL-1ß, IL-18, and IL-6 were significantly higher than arterial and venous levels (P=0.017, <0.001 and <0.001, respectively). Transcoronary (coronary sinus-arterial) gradients for IL-1ß, IL-18, and IL-6 were highest in ACS patients and lowest in controls (P=0.077, 0.033, and 0.014, respectively). Colchicine administration significantly reduced transcoronary gradients of all 3 cytokines in ACS patients by 40% to 88% (P=0.028, 0.032, and 0.032, for IL-1ß, IL-18, and IL-6, respectively). CONCLUSIONS: ACS patients exhibit increased local cardiac production of inflammatory cytokines. Short-term colchicine administration rapidly and significantly reduces levels of these cytokines.

MicroRNA-24 is a novel regulator of aldosterone and cortisol production in the human adrenal cortex.

Dysregulation of aldosterone or cortisol production can predispose to hypertension, as seen in aldosterone-producing adenoma, a form of primary aldosteronism. We investigated the role of microRNA (miRNA) in their production, with particular emphasis on the CYP11B1 (11ß-hydroxylase) and CYP11B2 (aldosterone synthase) genes, which produce the enzymes responsible for the final stages of cortisol and aldosterone biosynthesis, respectively. Knockdown of Dicer1, a key enzyme in miRNA maturation, significantly altered CYP11B1 and CYP11B2 expression in a human adrenocortical cell line. Screening of nondiseased human adrenal and aldosterone-producing adenoma samples yielded reproducible but distinctive miRNA expression signatures for each tissue type, with levels of certain miRNA, including microRNA-24 (miR-24), differing significantly between the 2. Bioinformatic analysis identified putative binding sites for several miRNA, including miR-24, in the 3' untranslated region of CYP11B1 and CYP11B2 mRNAs. In vitro manipulation of miR-24 confirmed its ability to modulate CYP11B1 and CYP11B2 expression, as well as cortisol and aldosterone production. This study demonstrates that Dicer-dependent miRNA, including miR-24, can post-transcriptionally regulate expression of the CYP11B1 and CYP11B2 genes. Normal adrenal tissue and aldosterone-producing adenoma differ significantly and reproducibly in their miRNA expression profiles, with miR-24 significantly downregulated in the latter. Adrenal miRNA may, therefore, be a novel and valid target for the therapeutic manipulation of corticosteroid biosynthesis.