Serine proteases mediate leukocyte recruitment and hepatic microvascular injury in the acute phase following extended hepatectomy.

OBJECTIVE: Post-hepatectomy liver failure (PHLF) is the main limitation of extended liver resection. The molecular mechanism and the role of leukocytes in the development of PHLF remain to be unveiled. We aimed to address the impact of serine proteases (SPs) on the acute phase after liver resection by intravitally analyzing leukocyte recruitment and changes in hemodynamics and microcirculation of the liver. METHODS: C57BL/6 mice undergoing 60% partial hepatectomy were treated with aprotinin (broad-spectrum SP inhibitor), tranexamic acid (plasmin inhibitor), or vehicle. Sham-operated animals served as controls. In vivo fluorescence microscopy was used to quantify leukocyte-endothelial interactions immediately after, as well as 120 min after partial hepatectomy in postsinusoidal venules, along with measurement of sinusoidal perfusion rate and postsinusoidal shear rate. Recruitment of leukocytes, neutrophils, T cells, and parameters of liver injury were assessed in tissue/blood samples. RESULTS: Leukocyte recruitment, sinusoidal perfusion failure rate, and shear rate were significantly increased in mice after 60% partial hepatectomy compared to sham-operated animals. The inhibition of SPs or plasmin significantly attenuated leukocyte recruitment and improved the perfusion rate in the remnant liver. ICAM-1 expression and neutrophil recruitment significantly increased after 60% partial hepatectomy and were strongly reduced by plasmin inhibition. CONCLUSIONS: Endothelial activation and leukocyte recruitment in the liver in response to the increment of sinusoidal shear rate were hallmarks in the acute phase after liver resection. SPs mediated leukocyte recruitment and contributed to the impairment of sinusoidal perfusion in an ICAM-1-dependent manner in the acute phase after liver resection.

Long noncoding RNAs in cardiovascular disease.

PURPOSE OF REVIEW: Here, we review recent findings on the role of long noncoding RNAs (lncRNAs) in cardiovascular disease (CVD). In addition, we highlight some of the latest findings in lncRNA biology, providing an outlook for future avenues of lncRNA research in CVD. RECENT FINDINGS: Recent publications provide translational evidence from patient studies and animal models for the role of specific lncRNAs in CVD. The molecular effector mechanisms of these lncRNAs are diverse. Overall, cell-type selective modulation of gene expression is the largest common denominator. New methods, such as single-cell profiling and CRISPR/Cas9-screening, reveal additional novel mechanistic principles: For example, many lncRNAs establish RNA-based spatial compartments that concentrate effector proteins. Also, RNA modifications and splicing features can be determinants of lncRNA function. SUMMARY: lncRNA research is passing the stage of enumerating lncRNAs or recording simplified on-off expression switches. Mechanistic analyses are starting to reveal overarching principles of how lncRNAs can function. Exploring these principles with decisive genetic testing in vivo remains the ultimate test to discern how lncRNA loci, by RNA motifs or DNA elements, affect CVD pathophysiology.

Detection of a Parkinson's Disease-Specific MicroRNA Signature in Nasal and Oral Swabs.

BACKGROUND: Biomaterials from oral and nasal swabs provide, in theory, a potential resource for biomarker development. However, their diagnostic value has not yet been investigated in the context of Parkinson's disease (PD) and associated conditions. OBJECTIVE: We have previously identified a PD-specific microRNA (miRNA) signature in gut biopsies. In this work, we aimed to investigate the expression of miRNAs in routine buccal (oral) and nasal swabs obtained from cases with idiopathic PD and isolated rapid eye movement sleep behavior disorder (iRBD), a prodromal symptom that often precedes α-synucleinopathies. We aimed to address their value as a diagnostic biomarker for PD and their mechanistic contribution to PD onset and progression. METHODS: Healthy control cases (n = 28), cases with PD (n = 29), and cases with iRBD (n = 8) were prospectively recruited to undergo routine buccal and nasal swabs. Total RNA was extracted from the swab material, and the expression of a predefined set of miRNAs was quantified by quantitative real-time polymerase chain reaction. RESULTS: Statistical analysis revealed a significantly increased expression of hsa-miR-1260a in cases who had PD. Interestingly, hsa-miR-1260a expression levels correlated with diseases severity, as well as olfactory function, in the PD and iRBD cohorts. Mechanistically, hsa-miR-1260a segregated to Golgi-associated cellular processes with a potential role in mucosal plasma cells. Predicted hsa-miR-1260a target gene expression was reduced in iRBD and PD groups. CONCLUSIONS: Our work demonstrates oral and nasal swabs as a valuable biomarker pool in PD and associated neurodegenerative conditions. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Non-Invasive Zinc Protoporphyrin Screening Offers Opportunities for Secondary Prevention of Iron Deficiency in Blood Donors.

Background: Frequent blood donors are at high risk of developing iron deficiency. Currently, there is no potent screening during blood donation to detect iron deficient erythropoiesis (IDE) before anemia develops and deferral from donation is inevitable. Study Design and Methods: In addition to capillary and venous hemoglobin, the iron status of 99 frequent blood donors was assessed by various venous blood parameters and zinc protoporphyrin IX (ZnPP). ZnPP was determined by high-performance liquid chromatography (HPLC) and a new prototype fiber-optic device was employed for non-invasive measurements of ZnPP through the blood collection tubing (NI-tubing) and on lip tissue (NI-lip). We aimed to evaluate the feasibility and diagnostic value of the NI-tubing measurement for early detection of severe iron deficiency in blood donors. Results: NI-tubing and HPLC reference measurements of ZnPP showed narrow limits of agreement of 12.2 µmol ZnPP/mol heme and very high correlation (Spearman's Rho = 0.938). Using a cutoff of 65 µmol ZnPP/mol heme, NI-tubing measurements (n = 93) identified 100% of donors with iron deficiency anemia (IDA) and an additional 38% of donors with IDE. Accordingly, NI-tubing measurements would allow detection and selective protection of particularly vulnerable donors. Conclusion: NI-tubing measurements are an accurate and simple method to implement ZnPP determination into the routine blood donation process. ZnPP was able to identify the majority of subjects with IDE and IDA and might therefore be a valuable tool to provide qualified information to donors about dietary measures and adjustments of the donation interval and thereby help to prevent IDA and hemoglobin deferral in the future.

Sequential Defects in Cardiac Lineage Commitment and Maturation Cause Hypoplastic Left Heart Syndrome.

BACKGROUND: Complex molecular programs in specific cell lineages govern human heart development. Hypoplastic left heart syndrome (HLHS) is the most common and severe manifestation within the spectrum of left ventricular outflow tract obstruction defects occurring in association with ventricular hypoplasia. The pathogenesis of HLHS is unknown, but hemodynamic disturbances are assumed to play a prominent role. METHODS: To identify perturbations in gene programs controlling ventricular muscle lineage development in HLHS, we performed whole-exome sequencing of 87 HLHS parent-offspring trios, nuclear transcriptomics of cardiomyocytes from ventricles of 4 patients with HLHS and 15 controls at different stages of heart development, single cell RNA sequencing, and 3D modeling in induced pluripotent stem cells from 3 patients with HLHS and 3 controls. RESULTS: Gene set enrichment and protein network analyses of damaging de novo mutations and dysregulated genes from ventricles of patients with HLHS suggested alterations in specific gene programs and cellular processes critical during fetal ventricular cardiogenesis, including cell cycle and cardiomyocyte maturation. Single-cell and 3D modeling with induced pluripotent stem cells demonstrated intrinsic defects in the cell cycle/unfolded protein response/autophagy hub resulting in disrupted differentiation of early cardiac progenitor lineages leading to defective cardiomyocyte subtype differentiation/maturation in HLHS. Premature cell cycle exit of ventricular cardiomyocytes from patients with HLHS prevented normal tissue responses to developmental signals for growth, leading to multinucleation/polyploidy, accumulation of DNA damage, and exacerbated apoptosis, all potential drivers of left ventricular hypoplasia in absence of hemodynamic cues. CONCLUSIONS: Our results highlight that despite genetic heterogeneity in HLHS, many mutations converge on sequential cellular processes primarily driving cardiac myogenesis, suggesting novel therapeutic approaches.

Uncovering the molecular identity of cardiosphere-derived cells (CDCs) by single-cell RNA sequencing.

Cardiosphere-derived cells (CDCs) generated from human cardiac biopsies have been shown to have disease-modifying bioactivity in clinical trials. Paradoxically, CDCs' cellular origin in the heart remains elusive. We studied the molecular identity of CDCs using single-cell RNA sequencing (sc-RNAseq) in comparison to cardiac non-myocyte and non-hematopoietic cells (cardiac fibroblasts/CFs, smooth muscle cells/SMCs and endothelial cells/ECs). We identified CDCs as a distinct and mitochondria-rich cell type that shared biological similarities with non-myocyte cells but not with cardiac progenitor cells derived from human-induced pluripotent stem cells. CXCL6 emerged as a new specific marker for CDCs. By analysis of sc-RNAseq data from human right atrial biopsies in comparison with CDCs we uncovered transcriptomic similarities between CDCs and CFs. By direct comparison of infant and adult CDC sc-RNAseq data, infant CDCs revealed GO-terms associated with cardiac development. To analyze the beneficial effects of CDCs (pro-angiogenic, anti-fibrotic, anti-apoptotic), we performed functional in vitro assays with CDC-derived extracellular vesicles (EVs). CDC EVs augmented in vitro angiogenesis and did not stimulate scarring. They also reduced the expression of pro-apoptotic Bax in NRCMs. In conclusion, CDCs were disclosed as mitochondria-rich cells with unique properties but also with similarities to right atrial CFs. CDCs displayed highly proliferative, secretory and immunomodulatory properties, characteristics that can also be found in activated or inflammatory cell types. By special culture conditions, CDCs earn some bioactivities, including angiogenic potential, which might modify disease in certain disorders.

Short-Chain Fatty Acids Improve Poststroke Recovery via Immunological Mechanisms.

Recovery after stroke is a multicellular process encompassing neurons, resident immune cells, and brain-invading cells. Stroke alters the gut microbiome, which in turn has considerable impact on stroke outcome. However, the mechanisms underlying gut-brain interaction and implications for long-term recovery are largely elusive. Here, we tested the hypothesis that short-chain fatty acids (SCFAs), key bioactive microbial metabolites, are the missing link along the gut-brain axis and might be able to modulate recovery after experimental stroke. SCFA supplementation in the drinking water of male mice significantly improved recovery of affected limb motor function. Using in vivo wide-field calcium imaging, we observed that SCFAs induced altered contralesional cortex connectivity. This was associated with SCFA-dependent changes in spine and synapse densities. RNA sequencing of the forebrain cortex indicated a potential involvement of microglial cells in contributing to the structural and functional remodeling. Further analyses confirmed a substantial impact of SCFAs on microglial activation, which depended on the recruitment of T cells to the infarcted brain. Our findings identified that microbiota-derived SCFAs modulate poststroke recovery via effects on systemic and brain resident immune cells.SIGNIFICANCE STATEMENT Previous studies have shown a bidirectional communication along the gut-brain axis after stroke. Stroke alters the gut microbiota composition, and in turn, microbiota dysbiosis has a substantial impact on stroke outcome by modulating the immune response. However, until now, the mediators derived from the gut microbiome affecting the gut-immune-brain axis and the molecular mechanisms involved in this process were unknown. Here, we demonstrate that short-chain fatty acids, fermentation products of the gut microbiome, are potent and proregenerative modulators of poststroke neuronal plasticity at various structural levels. We identified that this effect was mediated via circulating lymphocytes on microglial activation. These results identify short-chain fatty acids as a missing link along the gut-brain axis and as a potential therapeutic to improve recovery after stroke.

Circulating Metabolites Differentiate Acute Ischemic Stroke from Stroke Mimics.

OBJECTIVE: Early discrimination of patients with ischemic stroke (IS) from stroke mimics (SMs) poses a diagnostic challenge. The circulating metabolome might reflect pathophysiological events related to acute IS. Here, we investigated the utility of early metabolic changes for differentiating IS from SM. METHODS: We performed untargeted metabolomics on serum samples obtained from patients with IS (N = 508) and SM (N = 349; defined by absence of a diffusion weighted imaging [DWI] positive lesion on magnetic resonance imaging [MRI]) who presented to the hospital within 24 hours after symptom onset (median time from symptom onset to blood sampling = 3.3 hours; interquartile range [IQR] = 1.6-6.7 hours) and from neurologically normal controls (NCs; N = 112). We compared diagnostic groups in a discovery-validation approach by applying multivariable linear regression models, machine learning techniques, and propensity score matching. We further performed a targeted look-up of published metabolite sets. RESULTS: Levels of 41 metabolites were significantly associated with IS compared to NCs. The top metabolites showing the highest value in separating IS from SMs were asymmetrical and symmetrical dimethylarginine, pregnenolone sulfate, and adenosine. Together, these 4 metabolites differentiated patients with IS from SMs with an area under the curve (AUC) of 0.90 in the replication sample, which was superior to multimodal cranial computed tomography (CT; AUC = 0.80) obtained for routine diagnostics. They were further superior to previously published metabolite sets detected in our samples. All 4 metabolites returned to control levels by day 90. INTERPRETATION: A set of 4 metabolites with known biological effects relevant to stroke pathophysiology shows unprecedented utility to identify patients with IS upon hospital arrival, thus encouraging further investigation, including multicenter studies. ANN NEUROL 2020;88:736-746.

Platelet reactivity and clinical outcomes in acute coronary syndrome patients treated with prasugrel and clopidogrel: a pre-specified exploratory analysis from the TROPICAL-ACS trial.

AIMS: The value of platelet function testing (PFT) in predicting clinical outcomes and guiding P2Y12-inhibitor treatment is uncertain. In a pre-specified sub-study of the TROPICAL-ACS trial, we assessed ischaemic and bleeding risks according to high platelet reactivity (HPR) and low platelet reactivity (LPR) to ADP in patients receiving uniform prasugrel vs. PFT-guided clopidogrel or prasugrel. METHODS AND RESULTS: Acute coronary syndrome patients with PFT done 14 days after hospital discharge were included with prior randomization to uniform prasugrel for 12 months (control group, no treatment modification) vs. early de-escalation from prasugrel to clopidogrel and PFT-guided maintenance treatment (HPR: switch-back to prasugrel, non-HPR: clopidogrel). The composite ischaemic endpoint included cardiovascular death, myocardial infarction, or stroke, while key safety outcome was Bleeding Academic Research Consortium (BARC) 2-5 bleeding, from PFT until 12 months. We identified 2527 patients with PFT results available: 1266 were randomized to the guided and 1261 to the control group. Before treatment adjustment, HPR was more prevalent in the guided group (40% vs. 15%), while LPR was more common in control patients (27% vs. 11%). Compared to control patients without HPR on prasugrel (n = 1073), similar outcomes were observed in guided patients kept on clopidogrel [n = 755, hazard ratio (HR): 1.06 (0.57-1.95), P = 0.86] and also in patients with HPR on clopidogrel switched to prasugrel [n = 511, HR: 0.96 (0.47-1.96), P = 0.91]. In contrast, HPR on prasugrel was associated with a higher risk for ischaemic events in control patients [n = 188, HR: 2.16 (1.01-4.65), P = 0.049]. Low platelet reactivity was an independent predictor of bleeding [HR: 1.74 (1.18-2.56), P = 0.005], without interaction (Pint = 0.76) between study groups. CONCLUSION: Based on this substudy of a randomized trial, selecting prasugrel or clopidogrel based on PFT resulted in similar ischaemic outcomes as uniform prasugrel therapy without HPR. Although infrequent, HPR on prasugrel was associated with increased risk of ischaemic events. Low platelet reactivity was a strong and independent predictor of bleeding both on prasugrel and clopidogrel.

IL-18 but Not IL-1 Signaling Is Pivotal for the Initiation of Liver Injury in Murine Non-Alcoholic Fatty Liver Disease.

Non-alcoholic fatty liver disease (NAFLD) is rising in prevalence, and a better pathophysiologic understanding of the transition to its inflammatory phenotype (NASH) is key to the development of effective therapies. To evaluate the contribution of the NLRP3 inflammasome and its downstream effectors IL-1 and IL-18 in this process, we applied the true-to-life "American lifestyle-induced obesity syndrome" (ALiOS) diet mouse model. Development of obesity, fatty liver and liver damage was investigated in mice fed for 24 weeks according to the ALiOS protocol. Lipidomic changes in mouse livers were compared to human NAFLD samples. Receptor knockout mice for IL-1 and IL-18 were used to dissect the impact of downstream signals of inflammasome activity on the development of NAFLD. The ALiOS diet induced obesity and liver steatosis. The lipidomic changes closely mimicked changes in human NAFLD. A pro-inflammatory gene expression pattern in liver tissue and increased serum liver transaminases indicated early liver damage in the absence of histological evidence of NASH. Mechanistically, Il-18r-/-- but not Il-1r-/- mice were protected from early liver damage, possibly due to silencing of the pro-inflammatory gene expression pattern. Our study identified NLRP3 activation and IL-18R-dependent signaling as potential modulators of early liver damage in NAFLD, preceding development of histologic NASH.

Non-invasive measurement of erythrocyte zinc protoporphyrin in children.

BACKGROUND: Reliable screening for iron deficiency (ID) has required a blood sample and cost-intensive laboratory measurements. A novel method to non-invasively measure erythrocyte zinc protoporphyrin (ZnPP), an established marker for ID, is evaluated in children. METHODS: ZnPP was determined non-invasively by fluorescence measurements on the wet vermillion of the lower lip in 99 hospitalized children aged 9 months to 5 years. For comparison, conventional ID parameters and ZnPP were determined from blood samples. RESULTS: The non-invasively measured ZnPP values had limits of agreement (LoA) of 14 µmol ZnPP/mol heme (95% confidence interval: 9-20) compared to fluorescence measurements directly in blood. Repeated high-performance liquid chromatography reference determinations had comparable LoA of 14 µmol ZnPP/mol heme (9-17). Non-invasive ZnPP measurements had sensitivity and specificity of 67% (39-88%) and 97% (91-99%), and negative and positive predictive value of 94% (90-97%) and 80% (55-93%), for detecting ID as defined by the soluble transferrin receptor (sTfR). In groups with more severe ID as defined by serum ferritin and sTfR, higher ZnPP values were found, with the highest ZnPP values for the group with ID anemia. CONCLUSION: Non-invasive ZnPP measurements are reliably feasible in children. The simple and fast method has the potential to enable wide-spread screening for ID.

RNA-Seq Identifies Circulating miR-125a-5p, miR-125b-5p, and miR-143-3p as Potential Biomarkers for Acute Ischemic Stroke.

RATIONALE: Currently, there are no blood-based biomarkers with clinical utility for acute ischemic stroke (IS). MicroRNAs show promise as disease markers because of their cell type-specific expression patterns and stability in peripheral blood. OBJECTIVE: To identify circulating microRNAs associated with acute IS, determine their temporal course up to 90 days post-stroke, and explore their utility as an early diagnostic marker. METHODS AND RESULTS: We used RNA sequencing to study expression changes of circulating microRNAs in a discovery sample of 20 patients with IS and 20 matched healthy control subjects. We further applied quantitative real-time polymerase chain reaction in independent samples for validation (40 patients with IS and 40 matched controls), replication (200 patients with IS, 100 healthy control subjects), and in 72 patients with transient ischemic attacks. Sampling of patient plasma was done immediately upon hospital arrival. We identified, validated, and replicated 3 differentially expressed microRNAs, which were upregulated in patients with IS compared with both healthy control subjects (miR-125a-5p [1.8-fold; P=1.5×10-6], miR-125b-5p [2.5-fold; P=5.6×10-6], and miR-143-3p [4.8-fold; P=7.8×10-9]) and patients with transient ischemic attack (miR-125a-5p: P=0.003; miR-125b-5p: P=0.003; miR-143-3p: P=0.005). Longitudinal analysis of expression levels up to 90 days after stroke revealed a normalization to control levels for miR-125b-5p and miR-143-3p starting at day 2 while miR-125a-5p remained elevated. Levels of all 3 microRNAs depended on platelet numbers in a platelet spike-in experiment but were unaffected by chemical hypoxia in Neuro2a cells and in experimental stroke models. In a random forest classification, miR-125a-5p, miR-125b-5p, and miR-143-3p differentiated between healthy control subjects and patients with IS with an area under the curve of 0.90 (sensitivity: 85.6%; specificity: 76.3%), which was superior to multimodal cranial computed tomography obtained for routine diagnostics (sensitivity: 72.5%) and previously reported biomarkers of acute IS (neuron-specific enolase: area under the curve=0.69; interleukin 6: area under the curve=0.82). CONCLUSIONS: A set of circulating microRNAs (miR-125a-5p, miR-125b-5p, and miR-143-3p) associates with acute IS and might have clinical utility as an early diagnostic marker.

Molecular roles and function of circular RNAs in eukaryotic cells.

Protein-coding and noncoding genes in eukaryotes are typically expressed as linear messenger RNAs, with exons arranged colinearly to their genomic order. Recent advances in sequencing and in mapping RNA reads to reference genomes have revealed that thousands of genes express also covalently closed circular RNAs. Many of these circRNAs are stable and contain exons, but are not translated into proteins. Here, we review the emerging understanding that both, circRNAs produced by co- and posttranscriptional head-to-tail "backsplicing" of a downstream splice donor to a more upstream splice acceptor, as well as circRNAs generated from intronic lariats during colinear splicing, may exhibit physiologically relevant regulatory functions in eukaryotes. We describe how circRNAs impact gene expression of their host gene locus by affecting transcriptional initiation and elongation or splicing, and how they partake in controlling the function of other molecules, for example by interacting with microRNAs and proteins. We conclude with an outlook how circRNA dysregulation affects disease, and how the stability of circRNAs might be exploited in biomedical applications.

Vascular CXCR4 Limits Atherosclerosis by Maintaining Arterial Integrity: Evidence From Mouse and Human Studies.

BACKGROUND: The CXCL12/CXCR4 chemokine ligand/receptor axis controls (progenitor) cell homeostasis and trafficking. So far, an atheroprotective role of CXCL12/CXCR4 has only been implied through pharmacological intervention, in particular, because the somatic deletion of the CXCR4 gene in mice is embryonically lethal. Moreover, cell-specific effects of CXCR4 in the arterial wall and underlying mechanisms remain elusive, prompting us to investigate the relevance of CXCR4 in vascular cell types for atheroprotection. METHODS: We examined the role of vascular CXCR4 in atherosclerosis and plaque composition by inducing an endothelial cell (BmxCreERT2-driven)-specific or smooth muscle cell (SMC, SmmhcCreERT2- or TaglnCre-driven)-specific deficiency of CXCR4 in an apolipoprotein E-deficient mouse model. To identify underlying mechanisms for effects of CXCR4, we studied endothelial permeability, intravital leukocyte adhesion, involvement of the Akt/WNT/ß-catenin signaling pathway and relevant phosphatases in VE-cadherin expression and function, vascular tone in aortic rings, cholesterol efflux from macrophages, and expression of SMC phenotypic markers. Finally, we analyzed associations of common genetic variants at the CXCR4 locus with the risk for coronary heart disease, along with CXCR4 transcript expression in human atherosclerotic plaques. RESULTS: The cell-specific deletion of CXCR4 in arterial endothelial cells (n=12-15) or SMCs (n=13-24) markedly increased atherosclerotic lesion formation in hyperlipidemic mice. Endothelial barrier function was promoted by CXCL12/CXCR4, which triggered Akt/WNT/ß-catenin signaling to drive VE-cadherin expression and stabilized junctional VE-cadherin complexes through associated phosphatases. Conversely, endothelial CXCR4 deficiency caused arterial leakage and inflammatory leukocyte recruitment during atherogenesis. In arterial SMCs, CXCR4 sustained normal vascular reactivity and contractile responses, whereas CXCR4 deficiency favored a synthetic phenotype, the occurrence of macrophage-like SMCs in the lesions, and impaired cholesterol efflux. Regression analyses in humans (n=259 796) identified the C-allele at rs2322864 within the CXCR4 locus to be associated with increased risk for coronary heart disease. In line, C/C risk genotype carriers showed reduced CXCR4 expression in carotid artery plaques (n=188), which was furthermore associated with symptomatic disease. CONCLUSIONS: Our data clearly establish that vascular CXCR4 limits atherosclerosis by maintaining arterial integrity, preserving endothelial barrier function, and a normal contractile SMC phenotype. Enhancing these beneficial functions of arterial CXCR4 by selective modulators might open novel therapeutic options in atherosclerosis.

Late-pregnancy dysglycemia in obese pregnancies after negative testing for gestational diabetes and risk of future childhood overweight: An interim analysis from a longitudinal mother-child cohort study.

BACKGROUND: Maternal pre-conception obesity is a strong risk factor for childhood overweight. However, prenatal mechanisms and their effects in susceptible gestational periods that contribute to this risk are not well understood. We aimed to assess the impact of late-pregnancy dysglycemia in obese pregnancies with negative testing for gestational diabetes mellitus (GDM) on long-term mother-child outcomes. METHODS AND FINDINGS: The prospective cohort study Programming of Enhanced Adiposity Risk in Childhood-Early Screening (PEACHES) (n = 1,671) enrolled obese and normal weight mothers from August 2010 to December 2015 with trimester-specific data on glucose metabolism including GDM status at the end of the second trimester and maternal glycated hemoglobin (HbA1c) at delivery as a marker for late-pregnancy dysglycemia (HbA1c ≥ 5.7% [39 mmol/mol]). We assessed offspring short- and long-term outcomes up to 4 years, and maternal glucose metabolism 3.5 years postpartum. Multivariable linear and log-binomial regression with effects presented as mean increments (Δ) or relative risks (RRs) with 95% confidence intervals (CIs) were used to examine the association between late-pregnancy dysglycemia and outcomes. Linear mixed-effects models were used to study the longitudinal development of offspring body mass index (BMI) z-scores. The contribution of late-pregnancy dysglycemia to the association between maternal pre-conception obesity and offspring BMI was estimated using mediation analysis. In all, 898 mother-child pairs were included in this unplanned interim analysis. Among obese mothers with negative testing for GDM (n = 448), those with late-pregnancy dysglycemia (n = 135, 30.1%) had higher proportions of excessive total gestational weight gain (GWG), excessive third-trimester GWG, and offspring with large-for-gestational-age birth weight than those without. Besides higher birth weight (Δ 192 g, 95% CI 100-284) and cord-blood C-peptide concentration (Δ 0.10 ng/ml, 95% CI 0.02-0.17), offspring of these women had greater weight gain during early childhood (Δ BMI z-score per year 0.18, 95% CI 0.06-0.30, n = 262) and higher BMI z-score at 4 years (Δ 0.58, 95% CI 0.18-0.99, n = 43) than offspring of the obese, GDM-negative mothers with normal HbA1c values at delivery. Late-pregnancy dysglycemia in GDM-negative mothers accounted for about one-quarter of the association of maternal obesity with offspring BMI at age 4 years (n = 151). In contrast, childhood BMI z-scores were not affected by a diagnosis of GDM in obese pregnancies (GDM-positive: 0.58, 95% CI 0.36-0.79, versus GDM-negative: 0.62, 95% CI 0.44-0.79). One mechanism triggering late-pregnancy dysglycemia in obese, GDM-negative mothers was related to excessive third-trimester weight gain (RR 1.72, 95% CI 1.12-2.65). Furthermore, in the maternal population, we found a 4-fold (RR 4.01, 95% CI 1.97-8.17) increased risk of future prediabetes or diabetes if obese, GDM-negative women had a high versus normal HbA1c at delivery (absolute risk: 43.2% versus 10.5%). There is a potential for misclassification bias as the predominantly used GDM test procedure changed over the enrollment period. Further studies are required to validate the findings and elucidate the possible third-trimester factors contributing to future mother-child health status. CONCLUSIONS: Findings from this interim analysis suggest that offspring of obese mothers treated because of a diagnosis of GDM appeared to have a better BMI outcome in childhood than those of obese mothers who-following negative GDM testing-remained untreated in the last trimester and developed dysglycemia. Late-pregnancy dysglycemia related to uncontrolled weight gain may contribute to the development of child overweight and maternal diabetes. Our data suggest that negative GDM testing in obese pregnancies is not an "all-clear signal" and should not lead to reduced attention and risk awareness of physicians and obese women. Effective strategies are needed to maintain third-trimester glycemic and weight gain control among otherwise healthy obese pregnant women.

Guided de-escalation of antiplatelet treatment in patients with acute coronary syndrome undergoing percutaneous coronary intervention (TROPICAL-ACS): a randomised, open-label, multicentre trial.

BACKGROUND: Current guidelines recommend potent platelet inhibition with prasugrel or ticagrelor for 12 months after an acute coronary syndrome managed with percutaneous coronary intervention (PCI). However, the greatest anti-ischaemic benefit of potent antiplatelet drugs over the less potent clopidogrel occurs early, while most excess bleeding events arise during chronic treatment. Hence, a stage-adapted treatment with potent platelet inhibition in the acute phase and de-escalation to clopidogrel in the maintenance phase could be an alternative approach. We aimed to investigate the safety and efficacy of early de-escalation of antiplatelet treatment from prasugrel to clopidogrel guided by platelet function testing (PFT). METHODS: In this investigator-initiated, randomised, open-label, assessor-blinded, multicentre trial (TROPICAL-ACS) done at 33 sites in Europe, patients were enrolled if they had biomarker-positive acute coronary syndrome with successful PCI and a planned duration of dual antiplatelet treatment of 12 months. Enrolled patients were randomly assigned (1:1) using an internet-based randomisation procedure with a computer-generated block randomisation with stratification across study sites to either standard treatment with prasugrel for 12 months (control group) or a step-down regimen (1 week prasugrel followed by 1 week clopidogrel and PFT-guided maintenance therapy with clopidogrel or prasugrel from day 14 after hospital discharge; guided de-escalation group). The assessors were masked to the treatment allocation. The primary endpoint was net clinical benefit (cardiovascular death, myocardial infarction, stroke or bleeding grade 2 or higher according to Bleeding Academic Research Consortium [BARC]) criteria) 1 year after randomisation (non-inferiority hypothesis; margin of 30%). Analysis was intention to treat. This study is registered with ClinicalTrials.gov, number NCT01959451, and EudraCT, 2013-001636-22. FINDINGS: Between Dec 2, 2013, and May 20, 2016, 2610 patients were assigned to study groups; 1304 to the guided de-escalation group and 1306 to the control group. The primary endpoint occurred in 95 patients (7%) in the guided de-escalation group and in 118 patients (9%) in the control group (pnon-inferiority=0·0004; hazard ratio [HR] 0·81 [95% CI 0·62-1·06], psuperiority=0·12). Despite early de-escalation, there was no increase in the combined risk of cardiovascular death, myocardial infarction, or stroke in the de-escalation group (32 patients [3%]) versus in the control group (42 patients [3%]; pnon-inferiority=0·0115). There were 64 BARC 2 or higher bleeding events (5%) in the de-escalation group versus 79 events (6%) in the control group (HR 0·82 [95% CI 0·59-1·13]; p=0·23). INTERPRETATION: Guided de-escalation of antiplatelet treatment was non-inferior to standard treatment with prasugrel at 1 year after PCI in terms of net clinical benefit. Our trial shows that early de-escalation of antiplatelet treatment can be considered as an alternative approach in patients with acute coronary syndrome managed with PCI. FUNDING: Klinikum der Universität München, Roche Diagnostics, Eli Lilly, and Daiichi Sankyo.

Revisiting biomarker discovery by plasma proteomics.

Clinical analysis of blood is the most widespread diagnostic procedure in medicine, and blood biomarkers are used to categorize patients and to support treatment decisions. However, existing biomarkers are far from comprehensive and often lack specificity and new ones are being developed at a very slow rate. As described in this review, mass spectrometry (MS)-based proteomics has become a powerful technology in biological research and it is now poised to allow the characterization of the plasma proteome in great depth. Previous "triangular strategies" aimed at discovering single biomarker candidates in small cohorts, followed by classical immunoassays in much larger validation cohorts. We propose a "rectangular" plasma proteome profiling strategy, in which the proteome patterns of large cohorts are correlated with their phenotypes in health and disease. Translating such concepts into clinical practice will require restructuring several aspects of diagnostic decision-making, and we discuss some first steps in this direction.

Plasma cystatin C is a predictor of renal dysfunction, acute-on-chronic liver failure, and mortality in patients with acutely decompensated liver cirrhosis.

The development of acute-on-chronic liver failure (ACLF) in patients with liver cirrhosis is associated with high mortality rates. Renal failure is the most significant organ dysfunction that occurs in ACLF. So far there are no biomarkers predicting ACLF. We investigated whether cystatin C (CysC) and neutrophil gelatinase-associated lipocalin (NGAL) can predict development of renal dysfunction (RD), hepatorenal syndrome (HRS), ACLF, and mortality. We determined the plasma levels of CysC and NGAL in 429 patients hospitalized for acute decompensation of cirrhosis in the EASL-CLIF Acute-on-Chronic Liver Failure in Cirrhosis (CANONIC) study. The patients were followed for 90 days. Patients without RD or ACLF at inclusion but with development of either had significantly higher baseline concentrations of CysC and NGAL compared to patients without. CysC, but not NGAL, was found to be predictive of RD (odds ratio, 9.4; 95% confidence interval [CI], 1.8-49.7), HRS (odds ratio, 4.2; 95% CI, 1.2-14.8), and ACLF (odds ratio, 5.9; 95% CI, 1.3-25.9). CysC at day 3 was not found to be a better predictor than baseline CysC. CysC and NGAL were both predictive of 90-day mortality, with hazard ratios for CysC of 3.1 (95% CI, 2.1-4.7) and for NGAL of 1.9 (95% CI, 1.5-2.4). CONCLUSION: Baseline CysC is a biomarker of RD, HRS, and ACLF and an independent predictor of mortality in patients with acutely decompensated liver cirrhosis, though determining CysC at day 3 did not provide any benefit; while NGAL is also associated with short-term mortality, it fails to predict development of RD, HRS, and ACLF. Baseline CysC may help to identify patients at risk earlier and improve clinical management. (Hepatology 2017;66:1232-1241).

Adventitial adipogenic degeneration is an unidentified contributor to aortic wall weakening in the abdominal aortic aneurysm.

OBJECTIVE: The processes driving human abdominal aortic aneurysm (AAA) progression are not fully understood. Although antiinflammatory and proteolytic strategies effectively quench aneurysm progression in preclinical models, so far all clinical interventions failed. These observations hint at an incomplete understanding of the processes involved in AAA progression and rupture. Interestingly, strong clinical and molecular associations exist between popliteal artery aneurysms (PAAs) and AAAs; however, PAAs have an extremely low propensity to rupture. We thus reasoned that differences between these aneurysms may provide clues toward (auxiliary) processes involved in AAA-related wall debilitation. A better understanding of the pathophysiologic processes driving AAA growth can contribute to pharmaceutical treatments in the future. METHODS: Aneurysmal wall samples were collected during open elective and emergency repair. Control perirenal aorta was obtained during kidney transplantation, and reference popliteal tissue obtained from the anatomy department. This study incorporates various techniques including (immuno)histochemistry, Western Blot, quantitative polymerase chain reaction, microarray, and cell culture. RESULTS: Histologic evaluation of AAAs, PAAs, and control aorta shows extensive medial (PAA) and transmural fibrosis (AAA), and reveals abundant adventitial adipocytes aggregates as an exclusive phenomenon of AAAs (P < .001). Quantitative polymerase chain reaction, immunohistochemistry, Western blotting, and microarray analysis showed enrichment of adipogenic mediators (C/EBP family P = .027; KLF5 P < .000; and peroxisome proliferator activated receptor-γ, P = .032) in AAA tissue. In vitro differentiation tests indicated a sharply increased adipogenic potential of AAA adventitial mesenchymal cells (P < .0001). Observed enrichment of adipocyte-related genes and pathways in ruptured AAA (P < .0003) supports an association between the extent of fatty degeneration and rupture. CONCLUSIONS: This translational study identifies extensive adventitial fatty degeneration as an ignored and distinctive feature of AAA disease. Enrichment of adipocyte genesis and adipocyte-related genes in ruptured AAA point to an association between the extent of fatty degeneration and rupture. This observation may (partly) explain the failure of medical therapy and could provide a lead for pharmaceutical alleviation of AAA progression.

CXCL8 hyper-signaling in the aortic abdominal aneurysm.

There are indications for elevated CXCL8 levels in abdominal aortic aneurysm disease (AAA). CXCL8 is concurrently involved in neutrophil-mediated inflammation and angiogenesis, two prominent and distinctive characteristics of AAA. As such we considered an evaluation of a role for CXCL8 in AAA progression relevant. ELISA's, real time PCR and array analysis were used to explore CXCL8 signaling in AAA wall samples. A role for CXCL8 in AAA disease was tested through the oral CXCR1/2 antagonist DF2156A in the elastase model of AAA disease. There is an extreme disparity in aortic wall CXCL8 content between AAA and aortic atherosclerotic disease (median [IQR] aortic wall CXCL8 content: 425 [141-1261] (AAA) vs. 23 [2.8-89] (atherosclerotic aorta) µg/g protein (P < 1 ·â€¯10-14)), and abundant expression of the CXCR1 and 2 receptors in AAA. Array analysis followed by pathway analysis showed that CXCL8 hyper-expression in AAA is followed increased by IL-8 signaling (Z-score for AAA vs. atherosclerotic control: 2.97, p < 0.0001). Interference with CXCL8 signaling through DF2156A fully abrogated AAA formation and prevented matrix degradation in the murine elastase model of AAA disease (p < 0.001). CXCL8-signaling is a prominent and distinctive feature of AAA, interference with the pathway constitutes a promising target for medical stabilization of AAA.