A Genetic Variant Associated with Five Vascular Diseases Is a Distal Regulator of Endothelin-1 Gene Expression.

Genome-wide association studies (GWASs) implicate the PHACTR1 locus (6p24) in risk for five vascular diseases, including coronary artery disease, migraine headache, cervical artery dissection, fibromuscular dysplasia, and hypertension. Through genetic fine mapping, we prioritized rs9349379, a common SNP in the third intron of the PHACTR1 gene, as the putative causal variant. Epigenomic data from human tissue revealed an enhancer signature at rs9349379 exclusively in aorta, suggesting a regulatory function for this SNP in the vasculature. CRISPR-edited stem cell-derived endothelial cells demonstrate rs9349379 regulates expression of endothelin 1 (EDN1), a gene located 600 kb upstream of PHACTR1. The known physiologic effects of EDN1 on the vasculature may explain the pattern of risk for the five associated diseases. Overall, these data illustrate the integration of genetic, phenotypic, and epigenetic analysis to identify the biologic mechanism by which a common, non-coding variant can distally regulate a gene and contribute to the pathogenesis of multiple vascular diseases.

Evolutionary trajectories of small cell lung cancer under therapy.

The evolutionary processes that underlie the marked sensitivity of small cell lung cancer (SCLC) to chemotherapy and rapid relapse are unknown1-3. Here we determined tumour phylogenies at diagnosis and throughout chemotherapy and immunotherapy by multiregion sequencing of 160 tumours from 65 patients. Treatment-naive SCLC exhibited clonal homogeneity at distinct tumour sites, whereas first-line platinum-based chemotherapy led to a burst in genomic intratumour heterogeneity and spatial clonal diversity. We observed branched evolution and a shift to ancestral clones underlying tumour relapse. Effective radio- or immunotherapy induced a re-expansion of founder clones with acquired genomic damage from first-line chemotherapy. Whereas TP53 and RB1 alterations were exclusively part of the common ancestor, MYC family amplifications were frequently not constituents of the founder clone. At relapse, emerging subclonal mutations affected key genes associated with SCLC biology, and tumours harbouring clonal CREBBP/EP300 alterations underwent genome duplications. Gene-damaging TP53 alterations and co-alterations of TP53 missense mutations with TP73, CREBBP/EP300 or FMN2 were significantly associated with shorter disease relapse following chemotherapy. In summary, we uncover key processes of the genomic evolution of SCLC under therapy, identify the common ancestor as the source of clonal diversity at relapse and show central genomic patterns associated with sensitivity and resistance to chemotherapy.

Machine Learning for Myocardial Infarction Compared With Guideline-Recommended Diagnostic Pathways.

BACKGROUND: Collaboration for the Diagnosis and Evaluation of Acute Coronary Syndrome (CoDE-ACS) is a validated clinical decision support tool that uses machine learning with or without serial cardiac troponin measurements at a flexible time point to calculate the probability of myocardial infarction (MI). How CoDE-ACS performs at different time points for serial measurement and compares with guideline-recommended diagnostic pathways that rely on fixed thresholds and time points is uncertain. METHODS: Patients with possible MI without ST-segment-elevation were enrolled at 12 sites in 5 countries and underwent serial high-sensitivity cardiac troponin I concentration measurement at 0, 1, and 2 hours. Diagnostic performance of the CoDE-ACS model at each time point was determined for index type 1 MI and the effectiveness of previously validated low- and high-probability scores compared with guideline-recommended European Society of Cardiology (ESC) 0/1-hour, ESC 0/2-hour, and High-STEACS (High-Sensitivity Troponin in the Evaluation of Patients With Suspected Acute Coronary Syndrome) pathways. RESULTS: In total, 4105 patients (mean age, 61 years [interquartile range, 50-74]; 32% women) were included, among whom 575 (14%) had type 1 MI. At presentation, CoDE-ACS identified 56% of patients as low probability, with a negative predictive value and sensitivity of 99.7% (95% CI, 99.5%-99.9%) and 99.0% (98.6%-99.2%), ruling out more patients than the ESC 0-hour and High-STEACS (25% and 35%) pathways. Incorporating a second cardiac troponin measurement, CoDE-ACS identified 65% or 68% of patients as low probability at 1 or 2 hours, for an identical negative predictive value of 99.7% (99.5%-99.9%); 19% or 18% as high probability, with a positive predictive value of 64.9% (63.5%-66.4%) and 68.8% (67.3%-70.1%); and 16% or 14% as intermediate probability. In comparison, after serial measurements, the ESC 0/1-hour, ESC 0/2-hour, and High-STEACS pathways identified 49%, 53%, and 71% of patients as low risk, with a negative predictive value of 100% (99.9%-100%), 100% (99.9%-100%), and 99.7% (99.5%-99.8%); and 20%, 19%, or 29% as high risk, with a positive predictive value of 61.5% (60.0%-63.0%), 65.8% (64.3%-67.2%), and 48.3% (46.8%-49.8%), resulting in 31%, 28%, or 0%, who require further observation in the emergency department, respectively. CONCLUSIONS: CoDE-ACS performs consistently irrespective of the timing of serial cardiac troponin measurement, identifying more patients as low probability with comparable performance to guideline-recommended pathways for MI. Whether care guided by probabilities can improve the early diagnosis of MI requires prospective evaluation. REGISTRATION: URL: https://www.clinicaltrials.gov; Unique identifier: NCT00470587.

Reduction of Z alpha-1 antitrypsin polymers in human iPSC-hepatocytes and mice by LRRK2 inhibitors.

BACKGROUND: Alpha-1 antitrypsin deficiency (A1ATD) is a life-threatening condition caused by the inheritance of the serpin family A member 1 "Z" genetic variant driving alpha-1 antitrypsin (AAT) protein misfolding in hepatocytes. There are no approved medicines for this disease. METHODS: We conducted a high-throughput image-based small molecule screen using patient-derived induced pluripotent stem cell-hepatocytes (iPSC-hepatocytes). Identified targets were validated in vitro using 3 independent patient iPSC lines. The effects of the identified target, leucine-rich repeat kinase 2 (LRRK2), were further evaluated in an animal model of A1ATD through histology and immunohistochemistry and in an autophagy-reporter line. Autophagy induction was assessed through immunoblot and immunofluorescence analyses. RESULTS: Small-molecule screen performed in iPSC-hepatocytes identified LRRK2 as a potentially new therapeutic target. Of the commercially available LRRK2 inhibitors tested, we identified CZC-25146, a candidate with favorable pharmacokinetic properties, as capable of reducing polymer load, increasing normal AAT secretion, and reducing inflammatory cytokines in both cells and PiZ mice. Mechanistically, this effect was achieved through the induction of autophagy. CONCLUSIONS: Our findings support the use of CZC-25146 and leucine-rich repeat kinase-2 inhibitors in hepatic proteinopathy research and their further investigation as novel therapeutic candidates for A1ATD.

Precise therapeutic gene correction by a simple nuclease-induced double-stranded break.

Current programmable nuclease-based methods (for example, CRISPR-Cas9) for the precise correction of a disease-causing genetic mutation harness the homology-directed repair pathway. However, this repair process requires the co-delivery of an exogenous DNA donor to recode the sequence and can be inefficient in many cell types. Here we show that disease-causing frameshift mutations that result from microduplications can be efficiently reverted to the wild-type sequence simply by generating a DNA double-stranded break near the centre of the duplication. We demonstrate this in patient-derived cell lines for two diseases: limb-girdle muscular dystrophy type 2G (LGMD2G)1 and Hermansky-Pudlak syndrome type 1 (HPS1)2. Clonal analysis of inducible pluripotent stem (iPS) cells from the LGMD2G cell line, which contains a mutation in TCAP, treated with the Streptococcus pyogenes Cas9 (SpCas9) nuclease revealed that about 80% contained at least one wild-type TCAP allele; this correction also restored TCAP expression in LGMD2G iPS cell-derived myotubes. SpCas9 also efficiently corrected the genotype of an HPS1 patient-derived B-lymphoblastoid cell line. Inhibition of polyADP-ribose polymerase 1 (PARP-1) suppressed the nuclease-mediated collapse of the microduplication to the wild-type sequence, confirming that precise correction is mediated by the microhomology-mediated end joining (MMEJ) pathway. Analysis of editing by SpCas9 and Lachnospiraceae bacterium ND2006 Cas12a (LbCas12a) at non-pathogenic 4-36-base-pair microduplications within the genome indicates that the correction strategy is broadly applicable to a wide range of microduplication lengths and can be initiated by a variety of nucleases. The simplicity, reliability and efficacy of this MMEJ-based therapeutic strategy should permit the development of nuclease-based gene correction therapies for a variety of diseases that are associated with microduplications.

Glycoprotein IIb/IIIa Inhibitors in Acute Myocardial Infarction and Angiographic Microvascular Obstruction: The REVERSE-FLOW Trial.

BACKGROUND AND AIMS: Glycoprotein (GP) IIb/IIIa inhibitors are recommended in acute myocardial infarction (AMI) for bailout treatment in case of angiographic microvascular obstruction (MVO), also termed no-reflow phenomenon, after percutaneous coronary intervention (PCI) with, however, lacking evidence (class IIa, level C). METHODS: The investigator-initiated, international, multicenter REVERSE-FLOW trial randomized 120 patients with AMI and Thrombolysis In Myocardial Infarction flow grade ≤2 after primary PCI to optimal medical therapy with or without GP IIb/IIIa inhibitor. The primary endpoint was infarct size (%LV) assessed by cardiac magnetic resonance (CMR). Secondary endpoints included CMR-derived MVO and 30-day adverse clinical events. The trial is registered with ClinicalTrials.gov: NCT02739711. RESULTS: The population was predominantly male (76.7%) with a median age of 66 years and ST-elevation myocardial infarction in 73.3% of patients. Clinical and angiographic characteristics were well balanced between the cohorts. Patients in the treatment group (n=62) received eptifibatide (n=41) or tirofiban (n=21). Infarct size assessed by CMR imaging was similar in both study groups (25.4% of left ventricular mass [LV] vs. 25.2%LV; p=0.386). However, the number of patients with evidence of CMR-derived MVO (74.5% vs. 92.2%; p=0.017) and the extent of MVO (2.1%LV vs. 3.4%LV; p=0.025) were significantly reduced in the GP IIb/IIIa inhibitor group compared to controls. Thirty-day outcome showed an increased bleeding risk after GP IIb/IIIa inhibitor administration restricted to non-life-threatening bleedings (22.6% vs. 6.9%; p=0.016) without differences in all-cause mortality (4.8% vs. 3.4%; p=0.703). CONCLUSIONS: Bailout GP IIb/IIIa inhibition in AMI patients with angiographic MVO failed to reduce the primary endpoint infarct size but decreased CMR-derived MVO and led to an increase in non-fatal bleeding events.

Artificial microRNA suppresses C9ORF72 variants and decreases toxic dipeptide repeat proteins in vivo.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that affects motor neurons, causing progressive muscle weakness and respiratory failure. The presence of an expanded hexanucleotide repeat in chromosome 9 open reading frame 72 (C9ORF72) is the most frequent mutation causing familial ALS and frontotemporal dementia (FTD). To determine if suppressing expression of C9ORF72 gene products can reduce toxicity, we designed a set of artificial microRNAs (amiRNA) targeting the human C9ORF72 gene. Here we report that an AAV9-mediated amiRNA significantly suppresses expression of the C9ORF72 mRNA, protein, and toxic dipeptide repeat proteins generated by the expanded repeat in the brain and spinal cord of C9ORF72 transgenic mice.

Citrullination, a novel posttranslational modification of elastin, is involved in COPD pathogenesis.

Elastin is an extracellular matrix protein (ECM) that supports elasticity of the lung, and in patients with chronic obstructive pulmonary disease (COPD) and emphysema, the structural changes that reduce the amount of elastic recoil, lead to loss of pulmonary function. We recently demonstrated that elastin is a target of peptidyl arginine deiminase (PAD) enzyme-induced citrullination, thereby leading to enhanced susceptibility of this ECM protein to proteolysis. This study aimed to investigate the impact of PAD activity in vivo and furthermore assessed whether pharmacological inhibition of PAD activity protects against pulmonary emphysema. Using a Serpina1a-e knockout mouse model, previously shown to develop inflammation-mediated emphysema, we validated the involvement of PADs in airway disease. In line with emphysema development, intratracheal administration of lipopolysaccharide in combination with PADs provoked significant airspace enlargement (P < 0.001) and diminished lung function, including loss of lung tissue elastance (P = 0.0217) and increases in lung volumes (P = 0.0463). Intraperitoneal treatment of mice with the PAD inhibitor, BB-Cl-amidine, prevented PAD/LPS-mediated lung function decline and emphysema and reduced levels of citrullinated airway elastin (P = 0.0199). These results provide evidence for the impact of PADs on lung function decline, indicating promising potential for the future development of PAD-based therapeutics for preserving lung function in patients with COPD.NEW & NOTEWORTHY This study provides evidence for the impact of peptidyl arginine deiminase (PAD) enzymes on lung function decline, indicating promising potential for the future development of PAD-based therapeutics for preserving lung function in patients with COPD.

Monocyte NLRP3 inflammasome and interleukin-1ß activation modulated by alpha-1 antitrypsin therapy in deficient individuals.

INTRODUCTION: Altered complement component 3 (C3) activation in patients with alpha-1 antitrypsin (AAT) deficiency (AATD) has been reported. To understand the potential impact on course of inflammation, the aim of this study was to investigate whether C3d, a cleavage-product of C3, triggers interleukin (IL)-1ß secretion via activation of NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome. The objective was to explore the effect of AAT augmentation therapy in patients with AATD on the C3d/complement receptor 3 (CR3) signalling axis of monocytes and on circulating pro-inflammatory markers. METHODS: Inflammatory mediators were detected in blood from patients with AATD (n=28) and patients with AATD receiving augmentation therapy (n=19). Inflammasome activation and IL-1ß secretion were measured in monocytes of patients with AATD, and following C3d stimulation in the presence or absence of CR3 or NLRP3 inhibitors. RESULTS: C3d acting via CR3 induces NLRP3 and pro-IL-1ß production, and through induction of endoplasmic reticulum (ER) stress and calcium flux, triggers caspase-1 activation and IL-1ß secretion. Treatment of individuals with AATD with AAT therapy results in decreased plasma levels of C3d (3.0±1.2 µg/mL vs 1.3±0.5 µg/mL respectively, p<0.0001) and IL-1ß (115.4±30 pg/mL vs 73.3±20 pg/mL, respectively, p<0.0001), with a 2.0-fold decrease in monocyte NLRP3 protein expression (p=0.0303), despite continued ER stress activation. DISCUSSION: These results provide strong insight into the mechanism of complement-driven inflammation associated with AATD. Although the described variance in C3d and NLRP3 activation decreased post AAT augmentation therapy, results demonstrate persistent C3d and monocyte ER stress, with implications for new therapeutics and clinical practice.

External validation of the 0/1h-algorithm and derivation of a 0/2h-algorithm using a new point-of-care Hs-cTnI assay.

BACKGROUND: The high-sensitivity cardiac troponin (hs-cTn) I point-of-care (POC) hs-cTnI-PATHFAST assay has recently become clinically available. METHODS: We aimed to externally validate the hs-cTnI-PATHFAST 0/1h-algorithm recently developed for the early diagnosis of non-ST-segment-elevation myocardial infarction (NSTEMI) and derive and validate a 0/2-algorithm in patients presenting to the emergency department with acute chest discomfort included in a multicenter diagnostic study. Two independent cardiologists centrally adjudicated the final diagnoses using all the clinical and study-specific information available including serial measurements of hs-cTnI-Architect. RESULTS: Among 1,532 patients (median age 60 years, 33% [n = 501] women), NSTEMI was the final diagnosis in 13%. External validation of the hs-cTnI-PATHFAST 0/1h-algorithm showed very high negative predictive value (NPV; 100% [95%CI, 99.5%-100%]) and sensitivity 100% (95%CI, 98.2%-100%) for rule-out of NSTEMI. Positive predictive value (PPV) and specificity for rule-in of NSTEMI were high (74.9% [95%CI, 68.3%-80.5%] and 96.4% [95%CI, 95.2%-97.3%], respectively). Among 1,207 patients (median age 61 years, 32% [n = 391] women) available for the derivation (n = 848) and validation (n = 359) of the hs-cTnI-PATHFAST 0/2h-algorithm, a 0h-concentration <3 ng/L or a 0h-concentration <4 ng/L with a 2h-delta <4ng/L ruled-out NSTEMI in 52% of patients with a NPV of 100% (95%CI, 98-100) and sensitivity of 100% (95%CI, 92.9%-100%) in the validation cohort. A 0h-concentration ≥90ng/L or a 2h-delta ≥ 55ng/L ruled-in 38 patients (11%): PPV 81.6% (95%CI, 66.6-90.8), specificity 97.7% (95%CI, 95.4-98.9%). CONCLUSIONS: The POC hs-cTnI-PATHFAST assay allows rapid and effective rule-out and rule-in of NSTEMI using both a 0/1h- and a 0/2h-algorithm with high NPV/sensitivity for rule-out and high PPV/specificity for rule-in. CLINICAL TRIAL REGISTRATION: NCT00470587.

Association of preoperative beta-blocker use and cardiac complications after major noncardiac surgery: a prospective cohort study.

INTRODUCTION: Cardiac complications after major noncardiac surgery are common and associated with high morbidity and mortality. How preoperative use of beta-blockers may impact perioperative cardiac complications remains unclear. METHODS: In a multicentre prospective cohort study, preoperative beta-blocker use was ascertained in consecutive patients at elevated cardiovascular risk undergoing major noncardiac surgery. Cardiac complications were prospectively monitored and centrally adjudicated by two independent experts. The primary endpoint was perioperative myocardial infarction or injury attributable to a cardiac cause (cardiac PMI) within the first three postoperative days. The secondary endpoints were major adverse cardiac events (MACE), defined as a composite of myocardial infarction, acute heart failure, life-threatening arrhythmia, and cardiovascular death and all-cause death after 365 days. We used inverse probability of treatment weighting to account for differences between patients receiving beta-blockers and those who did not. RESULTS: A total of 3839/10 272 (37.4%) patients (mean age 74 yr; 44.8% female) received beta-blockers before surgery. Patients on beta-blockers were older, and more likely to be male with established cardiorespiratory and chronic kidney disease. Cardiac PMI occurred in 1077 patients, with a weighted odds ratio of 1.03 (95% confidence interval [CI] 0.94-1.12, P=0.55) for patients on beta-blockers. Within 365 days of surgery, 971/10 272 (9.5%) MACE had occurred, with a weighted hazard ratio of 0.99 (95% CI 0.83-1.18, P=0.90) for patients on beta-blockers. CONCLUSION: Preoperative use of beta-blockers was not associated with decreased cardiac complications including cardiac perioperative myocardial infarction or injury and major adverse cardiac event. Additionally, preoperative use of beta-blockers was not associated with increased all-cause death within 30 and 365 days. CLINICAL TRIAL REGISTRATION: NCT02573532.

Perioperative copeptin: predictive value and risk stratification in patients undergoing major noncardiac surgery-a prospective observational cohort study.

PURPOSE: Biomarkers can aid in perioperative risk stratification. While preoperative copeptin has been associated with adverse events, intraoperative information is lacking and this association may rather reflect a baseline risk. Knowledge about correlations between postoperative copeptin measurements and clinically relevant outcomes is scarce. We examined the association of perioperative copeptin concentrations with postoperative all-cause mortality and/or major adverse cardiac and cerebrovascular events (MACCE) at 12 months and 30 days as well as with perioperative myocardial injury (PMI). METHODS: We conducted a prospective observational cohort study of adults undergoing noncardiac surgery with intermediate to high surgical risk in Basel, Switzerland, and Düsseldorf, Germany from February 2016 to December 2020. We measured copeptin and cardiac troponin before surgery, immediately after surgery (0 hr) and once between the second and fourth postoperative day (POD 2-4). RESULTS: A primary outcome event of a composite of all-cause mortality and/or MACCE at 12 months occurred in 48/502 patients (9.6%). Elevated preoperative copeptin (> 14 pmol·L-1), immediate postoperative copeptin (> 90 pmol·L-1), and copeptin on POD 2-4 (> 14 pmol·L-1) were associated with lower one-year MACCE-free and/or mortality-free survival (hazard ratio [HR], 2.89; 95% confidence interval [CI], 1.62 to 5.2; HR, 2.07; 95% CI, 1.17 to 3.66; and HR, 2.47; 95% CI, 1.36 to 4.46, respectively). Multivariable analysis continued to show an association for preoperative and postoperative copeptin on POD 2-4. Furthermore, elevated copeptin on POD 2-4 showed an association with 30-day MACCE-free survival (HR, 2.15; 95% CI, 1.18 to 3.91). A total of 64 of 489 patients showed PMI (13.1%). Elevated preoperative copeptin was not associated with PMI, while immediate postoperative copeptin was modestly associated with PMI. CONCLUSION: The results of the present prospective observational cohort study suggest that perioperative copeptin concentrations can help identify patients at risk for all-cause mortality and/or MACCE. Other identified risk factors were revised cardiac risk index, body mass index, surgical risk, and preoperative hemoglobin. TRIAL REGISTRATION: ClinicalTrials.gov (NCT02687776); first submitted 9 February 2016.

RéSUMé: OBJECTIF: Les biomarqueurs peuvent aider à la stratification du risque périopératoire. Bien que la copeptine préopératoire ait été associée à des événements indésirables, les informations peropératoires font défaut; plutôt, cette association pourrait refléter un risque de base. Les connaissances sur les corrélations entre les mesures postopératoires de la copeptine et les résultats cliniquement pertinents sont rares. Nous avons examiné l'association entre les concentrations de copeptine périopératoires et la mortalité postopératoire toutes causes confondues et/ou les événements indésirables cardiaques et cérébrovasculaires majeurs (EICCM/MACCE) à 12 mois et 30 jours ainsi qu'en cas de lésion myocardique périopératoire (LMP/PMI). MéTHODE: Nous avons réalisé une étude de cohorte observationnelle prospective d'adultes bénéficiant d'une chirurgie non cardiaque à risque chirurgical intermédiaire à élevé à Bâle, en Suisse, et à Düsseldorf, en Allemagne, de février 2016 à décembre 2020. Nous avons mesuré la copeptine et la troponine cardiaque avant la chirurgie, immédiatement après la chirurgie (0 h) et une fois entre le deuxième et le quatrième jour postopératoire (JPO 2-4). RéSULTATS: Un événement constituant un critère d'évaluation principal d'un composite de mortalité toutes causes confondues et/ou de MACCE à 12 mois est survenu chez 48/502 patient·es (9,6 %). Une élévation de la copeptine préopératoire (> 14 pmol·L−1), de la copeptine postopératoire immédiate (> 90 pmol·L−1) et de la copeptine aux JPO 2 à 4 (> 14 pmol·L−1) était associée à une survie sans MACCE et/ou sans mortalité à un an plus faible (rapport de risque [RR], 2,89; intervalle de confiance [IC] à 95 %, 1,62 à 5,2; RR, 2,07; IC 95 %, 1,17 à 3,66; et RR, 2,47; IC 95 %, 1,36 à 4,46, respectivement). L'analyse multivariée a aussi montré une association entre la copeptine préopératoire et postopératoire aux JPO 2 à 4. De plus, un taux élevé de copeptine aux JPO 2 à 4 a montré une association avec la survie sans MACCE à 30 jours (RR, 2,15; IC 95 %, 1,18 à 3,91). Au total, 64 des 489 patient·es présentaient une LMP (13,1 %). Un taux élevé de copeptine préopératoire n'a pas été associé à la LMP, tandis que la copeptine postopératoire immédiate était modestement associée à la LMP. CONCLUSION: Les résultats de la présente étude de cohorte observationnelle prospective suggèrent que les concentrations périopératoires de copeptine peuvent aider à identifier les personnes à risque de mortalité toutes causes confondues et/ou de MACCE. Les autres facteurs de risque identifiés étaient l'indice de risque cardiaque révisé, l'indice de masse corporelle, le risque chirurgical et l'hémoglobine préopératoire. ENREGISTREMENT DE L'éTUDE: ClinicalTrials.gov (NCT02687776); première soumission le 9 février 2016.

Hypoimmune induced pluripotent stem cell-derived cell therapeutics treat cardiovascular and pulmonary diseases in immunocompetent allogeneic mice.

The emerging field of regenerative cell therapy is still limited by the few cell types that can reliably be differentiated from pluripotent stem cells and by the immune hurdle of commercially scalable allogeneic cell therapeutics. Here, we show that gene-edited, immune-evasive cell grafts can survive and successfully treat diseases in immunocompetent, fully allogeneic recipients. Transplanted endothelial cells improved perfusion and increased the likelihood of limb preservation in mice with critical limb ischemia. Endothelial cell grafts transduced to express a transgene for alpha1-antitrypsin (A1AT) successfully restored physiologic A1AT serum levels in mice with genetic A1AT deficiency. This cell therapy prevented both structural and functional changes of emphysematous lung disease. A mixture of endothelial cells and cardiomyocytes was injected into infarcted mouse hearts, and both cell types orthotopically engrafted in the ischemic areas. Cell therapy led to an improvement in invasive hemodynamic heart failure parameters. Our study supports the development of hypoimmune, universal regenerative cell therapeutics for cost-effective treatments of major diseases.

Up-regulation of miR-34b/c by JNK and FOXO3 protects from liver fibrosis.

α1-Antitrypsin (AAT) deficiency is a common genetic disease presenting with lung and liver diseases. AAT deficiency results from pathogenic variants in the SERPINA1 gene encoding AAT and the common mutant Z allele of SERPINA1 encodes for Z α1-antitrypsin (ATZ), a protein forming hepatotoxic polymers retained in the endoplasmic reticulum of hepatocytes. PiZ mice express the human ATZ and are a valuable model to investigate the human liver disease of AAT deficiency. In this study, we investigated differential expression of microRNAs (miRNAs) between PiZ and control mice and found that miR-34b/c was up-regulated and its levels correlated with intrahepatic ATZ. Furthermore, in PiZ mouse livers, we found that Forkhead Box O3 (FOXO3) driving microRNA-34b/c (miR-34b/c) expression was activated and miR-34b/c expression was dependent upon c-Jun N-terminal kinase (JNK) phosphorylation on Ser574 Deletion of miR-34b/c in PiZ mice resulted in early development of liver fibrosis and increased signaling of platelet-derived growth factor (PDGF), a target of miR-34b/c. Activation of FOXO3 and increased miR-34c were confirmed in livers of humans with AAT deficiency. In addition, JNK-activated FOXO3 and miR-34b/c up-regulation were detected in several mouse models of liver fibrosis. This study reveals a pathway involved in liver fibrosis and potentially implicated in both genetic and acquired causes of hepatic fibrosis.

Troponin in early presenters to rule out myocardial infarction.

AIMS: Whether a single cardiac troponin measurement can safely rule out myocardial infarction in patients presenting within a few hours of symptom onset is uncertain. The study aim was to assess the performance of troponin in early presenters. METHODS AND RESULTS: In patients with possible myocardial infarction, the diagnostic performance of a single measurement of high-sensitivity cardiac troponin I at presentation was evaluated and externally validated in those tested ≤3, 4-12, and >12 h from symptom onset. The limit-of-detection (2 ng/L), rule-out (5 ng/L), and sex-specific 99th centile (16 ng/L in women; 34 ng/L in men) thresholds were compared. In 41 103 consecutive patients [60 (17) years, 46% women], 12 595 (31%) presented within 3 h, and 3728 (9%) had myocardial infarction. In those presenting ≤3 h, a threshold of 2 ng/L had greater sensitivity and negative predictive value [99.4% (95% confidence interval 99.2%-99.5%) and 99.7% (99.6%-99.8%)] compared with 5 ng/L [96.5% (96.2%-96.8%) and 99.3% (99.1%-99.4%)]. In those presenting ≥3 h, the sensitivity and negative predictive value were similar for both thresholds. The sensitivity of the 99th centile was low in early and late presenters at 71.4% (70.6%-72.2%) and 92.5% (92.0%-93.0%), respectively. Findings were consistent in an external validation cohort of 7088 patients. CONCLUSION: In early presenters, a single measurement of high-sensitivity cardiac troponin I below the limit of detection may facilitate the safe rule out of myocardial infarction. The 99th centile should not be used to rule out myocardial infarction at presentation even in those presenting later following symptom onset.

Long-term outcomes of perioperative myocardial infarction/injury after non-cardiac surgery.

AIMS: Perioperative myocardial infarction/injury (PMI) following non-cardiac surgery is a frequent cardiac complication. Better understanding of the underlying aetiologies and outcomes is urgently needed. METHODS AND RESULTS: Aetiologies of PMIs detected within an active surveillance and response programme were centrally adjudicated by two independent physicians based on all information obtained during clinically indicated PMI work-up including cardiac imaging among consecutive high-risk patients undergoing major non-cardiac surgery in a prospective multicentre study. PMI aetiologies were hierarchically classified into 'extra-cardiac' if caused by a primarily extra-cardiac disease such as severe sepsis or pulmonary embolism; and 'cardiac', further subtyped into type 1 myocardial infarction (T1MI), tachyarrhythmia, acute heart failure (AHF), or likely type 2 myocardial infarction (lT2MI). Major adverse cardiac events (MACEs) including acute myocardial infarction, AHF (both only from day 3 to avoid inclusion bias), life-threatening arrhythmia, and cardiovascular death as well as all-cause death were assessed during 1-year follow-up. Among 7754 patients (age 45-98 years, 45% women), PMI occurred in 1016 (13.1%). At least one MACE occurred in 684/7754 patients (8.8%) and 818/7754 patients died (10.5%) within 1 year. Outcomes differed starkly according to aetiology: in patients with extra-cardiac PMI, T1MI, tachyarrhythmia, AHF, and lT2MI 51%, 41%, 57%, 64%, and 25% had MACE, and 38%, 27%, 40%, 49%, and 17% patients died within 1 year, respectively, compared to 7% and 9% in patients without PMI. These associations persisted in multivariable analysis. CONCLUSION: At 1 year, most PMI aetiologies have unacceptably high rates of MACE and all-cause death, highlighting the urgent need for more intensive treatments. STUDY REGISTRATION: https://clinicaltrials.gov/ct2/show/NCT02573532.

Factors Associated With Persisting Olfactory Dysfunction After COVID-19.

BACKGROUND: Fortunately, the majority of COVID-19 patients recover from olfactory dysfunction (OD) within the first couple of weeks. However, from approximately 5% up to 20% continue to suffer from OD even more than 1 year after the onset. Nonetheless, factors associated with long-lasting OD are hardly known. The aim of this study was to identify favourable and disadvantageous markers of persisting OD in COVID-19 patients. METHODOLOGY: Sixty-six patients (46 female; mean age: 39.9 years) that suffer from OD longer than 6 months due to laboratory-confirmed SARS-CoV-2 infection have participated in this longitudinal study. Participants completed comprehensive psychophysical chemosensory tests (i.e., Sniffin' Sticks = TDI) and questionnaires twice at our department-on average 219 ± 80 (T-1) and 489 ± 89 (T-2) days after the onset of symptoms, respectively. Olfactory recovery rates were associated with demographic factors and questionnaires using linear regression analysis. RESULTS: Patients below 40 years of age improved better (TDI: 4.1 ± 4.3 vs. 0.7 ± 5.8; p = 0.008) and achieved statistically significant higher scores (TDI: 31.5 ± 4.0 vs. 27.3 ± 6.7; p = 0.033) regarding psychophysical chemosensory tests. Furthermore, linear regression analysis revealed that parosmia was associated with worse orthonasal smell function (T-1: ß = -0.346, p = 0.004; T-2: ß = -0.384, p = 0.001), especially concerning identification subtest (T-1: ß = -0.395, p = 0.001; T-2: ß = -0.398, p < 0.001). Moreover, increasing parosmia between T-1and T-2 led to worse orthonasal olfactory function (ß = -0.294, p = 0.016). CONCLUSIONS: Older age and parosmia seem to be unfavourable factors of persisting OD in COVID-19 patients.

Evaluating Novel Protein Phosphatase 2A Activators as Therapeutics for Emphysema.

The activity of PP2A (protein phosphatase 2A), a serine-threonine phosphatase, is reduced by chronic cigarette smoke (SM) exposure and α-1 antitrypsin (AAT) deficiency, and chemical activation of PP2A reduces the loss of lung function in SM-exposed mice. However, the previously studied PP2A-activator tricyclic sulfonamide compound DBK-1154 has low stability to oxidative metabolism, resulting in fast clearance and low systemic exposure. Here we compare the utility of a new more stable PP2A activator, ATUX-792, versus DBK-1154 for the treatment of SM-induced emphysema. ATUX-792 was also tested in human bronchial epithelial cells and a mouse model of AAT deficiency, Serpina1a-e-knockout mice. Human bronchial epithelial cells were treated with ATUX-792 or DBK-1154, and cell viability, PP2A activity, and MAP (mitogen-activated protein) kinase phosphorylation status were examined. Wild-type mice received vehicle, DBK-1154, or ATUX-792 orally in the last 2 months of 4 months of SM exposure, and 8-month-old Serpina1a-e-knockout mice received ATUX-792 daily for 4 months. Forced oscillation and expiratory measurements and histology analysis were performed. Treatment with ATUX-792 or DBK-1154 resulted in PP2A activation, reduced MAP kinase phosphorylation, immune cell infiltration, reduced airspace enlargements, and preserved lung function. Using protein arrays and multiplex assays, PP2A activation was observed to reduce AAT-deficient and SM-induced release of CXCL5, CCL17, and CXCL16 into the airways, which coincided with reduced neutrophil lung infiltration. Our study indicates that suppression of the PP2A activity in two models of emphysema could be restored by next-generation PP2A activators to impact lung function.

Skeletal Muscle Disorders: A Noncardiac Source of Cardiac Troponin T.

BACKGROUND: Cardiac troponin (cTn) T and cTnI are considered cardiac specific and equivalent in the diagnosis of acute myocardial infarction. Previous studies suggested rare skeletal myopathies as a noncardiac source of cTnT. We aimed to confirm the reliability/cardiac specificity of cTnT in patients with various skeletal muscle disorders (SMDs). METHODS: We prospectively enrolled patients presenting with muscular complaints (≥2 weeks) for elective evaluation in 4 hospitals in 2 countries. After a cardiac workup, patients were adjudicated into 3 predefined cardiac disease categories. Concentrations of cTnT/I and resulting cTnT/I mismatches were assessed with high-sensitivity (hs-) cTnT (hs-cTnT-Elecsys) and 3 hs-cTnI assays (hs-cTnI-Architect, hs-cTnI-Access, hs-cTnI-Vista) and compared with those of control subjects without SMD presenting with adjudicated noncardiac chest pain to the emergency department (n=3508; mean age, 55 years; 37% female). In patients with available skeletal muscle biopsies, TNNT/I1-3 mRNA differential gene expression was compared with biopsies obtained in control subjects without SMD. RESULTS: Among 211 patients (mean age, 57 years; 42% female), 108 (51%) were adjudicated to having no cardiac disease, 44 (21%) to having mild disease, and 59 (28%) to having severe cardiac disease. hs-cTnT/I concentrations significantly increased from patients with no to those with mild and severe cardiac disease for all assays (all P<0.001). hs-cTnT-Elecsys concentrations were significantly higher in patients with SMD versus control subjects (median, 16 ng/L [interquartile range (IQR), 7-32.5 ng/L] versus 5 ng/L [IQR, 3-9 ng/L]; P<0.001), whereas hs-cTnI concentrations were mostly similar (hs-cTnI-Architect, 2.5 ng/L [IQR, 1.2-6.2 ng/L] versus 2.9 ng/L [IQR, 1.8-5.0 ng/L]; hs-cTnI-Access, 3.3 ng/L [IQR, 2.4-6.1 ng/L] versus 2.7 ng/L [IQR, 1.6-5.0 ng/L]; and hs-cTnI-Vista, 7.4 ng/L [IQR, 5.2-13.4 ng/L] versus 7.5 ng/L [IQR, 6-10 ng/L]). hs-cTnT-Elecsys concentrations were above the upper limit of normal in 55% of patients with SMD versus 13% of control subjects (P<0.01). mRNA analyses in skeletal muscle biopsies (n=33), mostly (n=24) from individuals with noninflammatory myopathy and myositis, showed 8-fold upregulation of TNNT2, encoding cTnT (but none for TNNI3, encoding cTnI) versus control subjects (n=16, PWald<0.001); the expression correlated with pathological disease activity (R=0.59, Pt-statistic<0.001) and circulating hs-cTnT concentrations (R=0.26, Pt-statistic=0.031). CONCLUSIONS: In patients with active chronic SMD, elevations in cTnT concentrations are common and not attributable to cardiac disease in the majority. This was not observed for cTnI and may be explained in part by re-expression of cTnT in skeletal muscle. REGISTRATION: URL: https://www. CLINICALTRIALS: gov; Unique identifier: NCT03660969.

SOD1 Suppression with Adeno-Associated Virus and MicroRNA in Familial ALS.

Two patients with familial amyotrophic lateral sclerosis (ALS) and mutations in the gene encoding superoxide dismutase 1 (SOD1) were treated with a single intrathecal infusion of adeno-associated virus encoding a microRNA targeting SOD1. In Patient 1, SOD1 levels in spinal cord tissue as analyzed on autopsy were lower than corresponding levels in untreated patients with SOD1-mediated ALS and in healthy controls. Levels of SOD1 in cerebrospinal fluid were transiently and only slightly lower in Patient 1 but were not affected in Patient 2. In Patient 1, meningoradiculitis developed after the infusion; Patient 2 was pretreated with immunosuppressive drugs and did not have this complication. Patient 1 had transient improvement in the strength of his right leg, a measure that had been relatively stable throughout his disease course, but there was no change in his vital capacity. Patient 2 had stable scores on a composite measure of ALS function and a stable vital capacity during a 12-month period. This study showed that intrathecal microRNA can be used as a potential treatment for SOD1-mediated ALS.