O-glycan determinants regulate VWF trafficking to Weibel-Palade bodies.

ABSTRACT: von Willebrand factor (VWF) undergoes complex posttranslational modification within endothelial cells (ECs) before secretion. This includes significant N- and O-linked glycosylation. Previous studies have demonstrated that changes in N-linked glycan structures significantly influence VWF biosynthesis. In contrast, although abnormalities in VWF O-linked glycans (OLGs) have been associated with enhanced VWF clearance, their effect on VWF biosynthesis remains poorly explored. Herein, we report a novel role for OLG determinants in regulating VWF biosynthesis and trafficking within ECs. We demonstrate that alterations in OLGs (notably reduced terminal sialylation) lead to activation of the A1 domain of VWF within EC. In the presence of altered OLG, VWF multimerization is reduced and Weibel-Palade body (WPB) formation significantly impaired. Consistently, the amount of VWF secreted from WPB after EC activation was significantly reduced in the context of O-glycosylation inhibition. Finally, altered OLG on VWF not only reduced the amount of VWF secreted after EC activation but also affected its hemostatic efficacy. Notably, VWF secreted after WPB exocytosis consisted predominantly of low molecular weight multimers, and the length of tethered VWF string formation on the surface of activated ECs was significantly reduced. In conclusion, our data therefore support the hypothesis that alterations in O-glycosylation pathways directly affect VWF trafficking within human EC. These findings are interesting given that previous studies have reported altered OLG on plasma VWF (notably increased T-antigen expression) in patients with von Willebrand disease.

Endothelial cell activation, Weibel-Palade body secretion, and enhanced angiogenesis in severe COVID-19.

Background: Severe COVID-19 is associated with marked endothelial cell (EC) activation that plays a key role in immunothrombosis and pulmonary microvascular occlusion. However, the biological mechanisms through which SARS-CoV-2 causes EC activation and damage remain poorly defined. Objectives: We investigated EC activation in patients with acute COVID-19, and specifically focused on how proteins stored within Weibel-Palade bodies may impact key aspects of disease pathogenesis. Methods: Thirty-nine patients with confirmed COVID-19 were recruited. Weibel-Palade body biomarkers (von Willebrand factor [VWF], angiopoietin-2 [Angpt-2], and osteoprotegerin) and soluble thrombomodulin (sTM) levels were determined. In addition, EC activation and angiogenesis were assessed in the presence or absence of COVID-19 plasma incubation. Results: Markedly elevated plasma VWF antigen, Angpt-2, osteoprotegerin, and sTM levels were observed in patients with acute COVID-19. The increased levels of both sTM and Weibel-Palade body components (VWF, osteoprotegerin, and Angpt-2) correlated with COVID-19 severity. Incubation of COVID-19 plasma with ECs triggered enhanced VWF secretion and increased Angpt-2 expression, as well as significantly enhanced in vitro EC tube formation and angiogenesis. Conclusion: We propose that acute SARS-CoV-2 infection leads to a complex and multifactorial EC activation, progressive loss of thrombomodulin, and increased Angpt-2 expression, which collectively serve to promote a local proangiogenic state.

Inland Waterways and Population Health and Wellbeing: A Cross-Sectional Study of Waterway Users in the UK.

Natural environments, such as inland waterways (IWs), have been identified as a potential means to increase physical activity and promote health and wellbeing. However, further information on predictors of IW usage and their relationship with health and wellbeing outcomes is needed. Data were taken from the cross-sectional UK Waterways Engagement Monitor survey of waterway users (n = 21,537) in 2019/2020. Health outcome measures were life satisfaction, physical activity, and mental wellbeing. Visit frequency was an additional outcome measure. Both bivariate and multivariable associations between outcome measures and features of IWs were explored. The travel-cost method was used to estimate users' demand, expressed by travel costs to waterways. Multivariable models showed positive associations of frequent visits and use for recreational/leisure purposes with life satisfaction and physical activity. Rural visits were associated with higher life satisfaction than urban ones. Lower visit satisfaction negatively impacted life satisfaction and mental wellbeing. Visit frequency was influenced by individual characteristics and purpose of visit, including visits for exercise. Waterway visits were inversely associated with travel costs (IRR = 0.99, p-value ≤ 0.001), and there was greater demand elasticity for short distances (≤5 miles). Socioeconomic-related inequalities were present. Future policies could enhance frequent use of waterways and alleviate accessibility-related inequalities to improve population health outcomes.

Sustained VWF-ADAMTS-13 axis imbalance and endotheliopathy in long COVID syndrome is related to immune dysfunction.

BACKGROUND: Prolonged recovery is common after acute SARS-CoV-2 infection; however, the pathophysiological mechanisms underpinning Long COVID syndrome remain unknown. VWF/ADAMTS-13 imbalance, dysregulated angiogenesis, and immunothrombosis are hallmarks of acute COVID-19. We hypothesized that VWF/ADAMTS-13 imbalance persists in convalescence together with endothelial cell (EC) activation and angiogenic disturbance. Additionally, we postulate that ongoing immune cell dysfunction may be linked to sustained EC and coagulation activation. PATIENTS AND METHODS: Fifty patients were reviewed at a minimum of 6 weeks following acute COVID-19. ADAMTS-13, Weibel Palade Body (WPB) proteins, and angiogenesis-related proteins were assessed and clinical evaluation and immunophenotyping performed. Comparisons were made with healthy controls (n = 20) and acute COVID-19 patients (n = 36). RESULTS: ADAMTS-13 levels were reduced (p = 0.009) and the VWF-ADAMTS-13 ratio was increased in convalescence (p = 0.0004). Levels of platelet factor 4 (PF4), a putative protector of VWF, were also elevated (p = 0.0001). A non-significant increase in WPB proteins Angiopoietin-2 (Ang-2) and Osteoprotegerin (OPG) was observed in convalescent patients and WPB markers correlated with EC parameters. Enhanced expression of 21 angiogenesis-related proteins was observed in convalescent COVID-19. Finally, immunophenotyping revealed significantly elevated intermediate monocytes and activated CD4+ and CD8+ T cells in convalescence, which correlated with thrombin generation and endotheliopathy markers, respectively. CONCLUSION: Our data provide insights into sustained EC activation, dysregulated angiogenesis, and VWF/ADAMTS-13 axis imbalance in convalescent COVID-19. In keeping with the pivotal role of immunothrombosis in acute COVID-19, our findings support the hypothesis that abnormal T cell and monocyte populations may be important in the context of persistent EC activation and hemostatic dysfunction during convalescence.

Comparison of four commercial solid-phase micro-extraction (SPME) fibres for the headspace characterisation and profiling of gunshot exhausts in spent cartridge casings.

Headspace solid-phase micro-extraction (SPME) is a promising technique for the characterisation and profiling of gunshot exhausts in spent cartridge casings, especially for health and environmental risk assessments, as well as forensic purposes. To date, however, no comprehensive investigation has been carried out to objectively assess the kinds of compound released during a discharge that can be recovered by this approach, the selectivity of the main commercially available fibres, and their relative performances for the analysis of gunshot exhausts and the discrimination of different ammunition types. This study aimed to fill this gap. Gunshot exhausts in spent cartridge casings from four different ammunition types were analysed by GC-MS, after extraction with four different commercial fibres: 100 µm polydimethylsiloxane (PDMS), 85 µm polyacrylate (PA), 65 µm polydimethylsiloxane/divinylbenzene (DVB), and 85 µm carboxen/polydimethylsiloxane (CAR). Results showed that, overall, a total of 120 analytes could be observed across the cartridges, but the different tested fibres also displayed distinct performances, which were, to some extent, complementary for the characterisation of gunshot exhausts. DVB, in particular, recovered the most compounds simultaneously. On the other hand, the observed variability between measurements was also high, making it a poor candidate for (semi-)quantitative applications (e.g. estimation of time since discharge and/or source profiling). In this regard, PA demonstrated the highest potential for broad use and implementation in multi-purpose methods.

High Number of Positive Rapid Plasma Reagin With Low Titers Using the Automated AIX 1000 RPR System.

ABSTRACT: This study evaluated the performance of manual rapid plasma reagin (RPR) and automated AIX1000 RPR tests for the diagnosis of syphilis. Manual RPR showed a 3% reactive result compared with 5.8% for the automated test mainly because of RPR reactive results with low titers not confirmed by Treponema pallidum particle agglutination.

Promoting Patient and Nurse Safety in Acute Dialysis Units Through Advocacy and Collaboration.

Hospitals face the increasing challenge of balancing the need to provide quality care with that of being cost-effective. While guidelines and benchmarks for quality care exist in many areas within the hospital, those guidelines can be lacking for the acute (inpatient) dialysis setting. Nephrology nurses working in this setting may provide hemodialysis, peritoneal dialysis, continuous kidney replacement therapy, and apheresis treatments at the patient's bedside and in an acute dialysis unit. This article discusses the need to promote patient and nurse safety in acute dialysis settings through advocacy and collaboration.

The HEART Pathway Randomized Controlled Trial One-year Outcomes.

OBJECTIVE: The objective was to determine the impact of the HEART Pathway on health care utilization and safety outcomes at 1 year in patients with acute chest pain. METHODS: Adult emergency department (ED) patients with chest pain (N = 282) were randomized to the HEART Pathway or usual care. In the HEART Pathway arm, ED providers used the HEART score and troponin measures (0 and 3 hours) to risk stratify patients. Usual care was based on American College of Cardiology/American Heart Association guidelines. Major adverse cardiac events (MACE-cardiac death, myocardial infarction [MI], or coronary revascularization), objective testing (stress testing or coronary angiography), and cardiac hospitalizations and ED visits were assessed at 1 year. Randomization arm outcomes were compared using Fisher's exact tests. RESULTS: A total of 282 patients were enrolled, with 141 randomized to each arm. MACE at 1 year occurred in 10.6% (30/282): 9.9% in the HEART Pathway arm (14/141; 10 MIs, four revascularizations without MI) versus 11.3% in usual care (16/141; one cardiac death, 13 MIs, two revascularizations without MI; p = 0.85). Among low-risk HEART Pathway patients, 0% (0/66) had MACE, with a negative predictive value (NPV) of 100% (95% confidence interval = 93%-100%). Objective testing through 1 year occurred in 63.1% (89/141) of HEART Pathway patients compared to 71.6% (101/141) in usual care (p = 0.16). Nonindex cardiac-related hospitalizations and ED visits occurred in 14.9% (21/141) and 21.3% (30/141) of patients in the HEART Pathway versus 10.6% (15/141) and 16.3% (23/141) in usual care (p = 0.37, p = 0.36). CONCLUSIONS: The HEART Pathway had a 100% NPV for 1-year safety outcomes (MACE) without increasing downstream hospitalizations or ED visits. Reduction in 1-year objective testing was not significant.

Adherence to an Accelerated Diagnostic Protocol for Chest Pain: Secondary Analysis of the HEART Pathway Randomized Trial.

OBJECTIVES: Accelerated diagnostic protocols (ADPs), such as the HEART Pathway, are gaining popularity in emergency departments (EDs) as tools used to risk stratify patients with acute chest pain. However, provider nonadherence may threaten the safety and effectiveness of ADPs. The objective of this study was to determine the frequency and impact of ADP nonadherence. METHODS: A secondary analysis of participants enrolled in the HEART Pathway RCT was conducted. This trial enrolled 282 adult ED patients with symptoms concerning for acute coronary syndrome without ST-elevation on electrocardiogram. Patients randomized to the HEART Pathway (N = 141) were included in this analysis. Outcomes included index visit disposition, nonadherence, and major adverse cardiac events (MACEs) at 30 days. MACE was defined as death, myocardial infarction, or revascularization. Nonadherence was defined as: 1) undertesting-discharging a high-risk patient from the ED without objective testing (stress testing or coronary angiography) or 2) overtesting-admitting or obtaining objective testing on a low-risk patient. RESULTS: Nonadherence to the HEART Pathway occurred in 28 of 141 patients (20%, 95% confidence interval [CI] = 14% to 27%). Overtesting occurred in 19 of 141 patients (13.5%, 95% CI = 8% to 19%) and undertesting in nine of 141 patients (6%, 95% CI = 3% to 12%). None of these 28 patients suffered MACE. The net effect of nonadherence was 10 additional admissions among patients identified as low-risk and appropriate for early discharge (absolute decrease in discharge rate of 7%, 95% CI = 3% to 13%). CONCLUSIONS: Real-time use of the HEART Pathway resulted in a nonadherence rate of 20%, mostly due to overtesting. None of these patients had MACE within 30 days. Nonadherence decreased the discharge rate, attenuating the HEART Pathway's impact on health care use.

Fertility after uterine artery embolization: a review.

Uterine artery embolization (UAE) research has largely been focused on women over 40 years, yet women of reproductive age undergo UAE without any increased morbidity. Some physicians refrain from recommending UAE to women in this age group because of some research findings showing a negative effect on fertility. This review presents a comprehensive discussion of the fertility potential of women undergoing UAE, in terms of pregnancy rates and complications as well as ovarian function and reserve. Findings indicate many benefits for women desiring fertility who undergo UAE over traditional myomectomy.

The HEART Pathway randomized trial: identifying emergency department patients with acute chest pain for early discharge.

BACKGROUND: The HEART Pathway is a decision aid designed to identify emergency department patients with acute chest pain for early discharge. No randomized trials have compared the HEART Pathway with usual care. METHODS AND RESULTS: Adult emergency department patients with symptoms related to acute coronary syndrome without ST-elevation on ECG (n=282) were randomized to the HEART Pathway or usual care. In the HEART Pathway arm, emergency department providers used the HEART score, a validated decision aid, and troponin measures at 0 and 3 hours to identify patients for early discharge. Usual care was based on American College of Cardiology/American Heart Association guidelines. The primary outcome, objective cardiac testing (stress testing or angiography), and secondary outcomes, index length of stay, early discharge, and major adverse cardiac events (death, myocardial infarction, or coronary revascularization), were assessed at 30 days by phone interview and record review. Participants had a mean age of 53 years, 16% had previous myocardial infarction, and 6% (95% confidence interval, 3.6%-9.5%) had major adverse cardiac events within 30 days of randomization. Compared with usual care, use of the HEART Pathway decreased objective cardiac testing at 30 days by 12.1% (68.8% versus 56.7%; P=0.048) and length of stay by 12 hours (9.9 versus 21.9 hours; P=0.013) and increased early discharges by 21.3% (39.7% versus 18.4%; P<0.001). No patients identified for early discharge had major adverse cardiac events within 30 days. CONCLUSIONS: The HEART Pathway reduces objective cardiac testing during 30 days, shortens length of stay, and increases early discharges. These important efficiency gains occurred without any patients identified for early discharge suffering MACE at 30 days. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique Identifier: NCT01665521.

Cloning of a functional 25-hydroxyvitamin D-1α-hydroxylase in zebrafish (Danio rerio).

Activation of precursor 25-hydroxyvitamin D3 (25D) to hormonal 1,25-dihydroxyvitamin D3 (1,25D) is a pivotal step in vitamin D physiology, catalysed by the enzyme 25-hydroxyvitamin D-1α-hydroxylase (1α-hydroxylase). To establish new models for assessing the physiological importance of the 1α-hydroxylase-25D-axis, we used Danio rerio (zebrafish) to characterize expression and biological activity of the gene for 1α-hydroxylase (cyp27b1). Treatment of day 5 zebrafish larvae with inactive 25D (5-150 nM) or active 1,25D (0.1-10 nM) induced dose responsive expression (15-95-fold) of the vitamin D-target gene cyp24a1 relative to larvae treated with vehicle, suggesting the presence of Cyp27b1 activity. A full-length zebrafish cyp27b1 cDNA was then generated using RACE and RT-PCR methods. Sequencing of the resulting clone revealed an open reading frame encoding a protein of 505 amino acids with 54% identity to human CYP27B1. Transfection of a cyp27b1 expression vector into HKC-8, a human kidney proximal tubular epithelial cell line, enhanced intracrine metabolism of 25D to 1,25D resulting in greater than twofold induction of CYP24A1 mRNA expression and a 25-fold increase in 1,25D production compared to empty vector. These data indicate that we have cloned a functional zebrafish CYP27B1, representing a phylogenetically distant branch from mammals of this key enzyme in vitamin D metabolism. Further analysis of cyp27b1 expression and activity in zebrafish may provide new perspectives on the biological importance of 25D metabolism.