Antithrombin testing and treatment in pregnancy: Their real-world relationship to clinical outcomes.

BACKGROUND: Antithrombin (AT) deficiency is a severe thrombophilia associated with increased rates of maternal morbidity, mortality, and greater healthcare resource utilization during pregnancy and postpartum. METHODS: Two large U.S. healthcare databases were queried for women aged 15-44 with delivery-related encounters: Cerner Real-World Data (CRWD, 01/01/2000-12/31/2021) and Premier Healthcare Database (PHD, 01/01/2016-01/01/2019). Individuals receiving cardiopulmonary bypass were excluded. Three cohorts were created: 1) Individuals who had AT levels tested any time between 9-months pre- through 3-months post-delivery (CRWD Test Cohort); 2) individuals prescribed AT concentrate (ATc) within 1-year pre- or 1-year post-delivery in CRWD (CRWD Medication Cohort); and 3) the same criteria as 2) applied to PHD (PHD Medication Cohort). RESULTS: There were 5411 individuals in the CRWD Test Cohort, 13 in the CRWD Medication Cohort and 38 in the PHD Medication Cohort. Demographic and baseline clinical characteristics were similar across cohorts. AT level testing occurred pre-delivery in 47.9 % of the CRWD Test Cohort and 23.1 % of the CRWD Medication Cohort. ATc was administered during the delivery hospitalization to 0.1 %, 23.1 % and 50.0 % of the CRWD Test, CRWD Medication, and PHD Medication Cohorts, respectively. Across cohorts, 5.4-7.9 % of individuals experienced thrombosis during the delivery-related encounter. Mean (SD) total costs for delivery through 1-year post-delivery were $190,894 ($276,893) with $123,763 ($177,122) of total costs related to abnormal coagulation. CONCLUSION: Opportunities exist to enhance the care of pregnant individuals with low AT levels throughout pregnancy, aiming for optimal maternal outcomes.

Performance comparison of 6 in-hospital patient monitoring systems in the detection and alarm of ventricular cardiac arrhythmias.

Background: Patient monitoring devices are critical for alerting of potential cardiac arrhythmias during hospitalization; however, there are concerns of alarm fatigue due to high false alarm rates. Objective: The purpose of this study was to evaluate the sensitivity and false alarm rate of hospital-based continuous electrocardiographic (ECG) monitoring technologies. Methods: Six commonly used multiparameter bedside monitoring systems available in the United States were evaluated: B125M (GE HealthCare), ePM10 and iPM12 (Mindray), Efficia and IntelliVue (Philips), and Life Scope (Nihon Kohden). Sensitivity was tested using ECG recordings containing 57 true ventricular tachycardia (VT) events. False-positive rate testing used 205 patient-hours of ECG recordings containing no cardiac arrhythmias. Signals from ECG recordings were fed to devices simultaneously; high-severity arrhythmia alarms were tracked. Sensitivity to true VT events and false-positive rates were determined. Differences were assessed using Fisher exact tests (sensitivity) and Z-tests (false-positive rates). Results: B125M raised 56 total alarms for 57 annotated VT events and had the highest sensitivity (98%; P <.05), followed by iPM12 (84%), Life Scope (81%), Efficia (79%), ePM10 (77%), and IntelliVue (75%). B125M raised 20 false alarms, which was significantly lower (P <.0001) than iPM12 (284), Life Scope (292), IntelliVue (304), ePM10 (324), and Efficia (493). The most common false alarm was VT, followed by nonsustained VT. Conclusion: We found significant performance differences among multiparameter bedside ECG monitoring systems using previously collected recordings. B125M had the highest sensitivity in detecting true VT events and lowest false alarm rate. These results can assist in minimizing alarm fatigue and optimizing patient safety by careful selection of in-hospital continuous monitoring technology.

Influence of wastewater treatment plants and water input sources on size, shape, and polymer distributions of microplastics in St. Andrew Bay, Florida, USA.

Microplastic (MP) pollution is an ongoing problem in coastal systems, where wastewater treatment plants (WWTPs) deposit particles daily. This study examined MP characteristics at WWTP outflow and control sites in St. Andrew Bay in Northwestern Florida, USA. WWTP sites contained mostly polypropylene fragments (180.1 µm avg. size), while reference sites contained polypropylene fragments, and polyethylene and polyester fibers (315.3 µm avg. size). MP sizes were strongly linked to distance from the nearest WWTP, while shape and polymer compositions were more closely related to dissolved oxygen concentrations and distance to the nearest water input source. The prevalence of polypropylene fragments at WWTP sites suggests that extreme weather events during the study flushed land-based debris into the system, where it was buried in the sediments. Increased abundances of polyester and polyethylene terephthalate in the winter at WWTP sites are indicative of the role that laundering synthetic textiles plays in coastal MP pollution.

Two-year retrospective cohort results on use of a dynamic unilateral brace for treatment of clubfoot: Can compliance and prevention of recurrence both be achieved?

Objectives: The Ponseti method has led to vast improvements in outcomes for infants born with clubfoot deformity, but challenges with compliance during the bracing phase of the protocol remain. Unilateral braces promise higher compliance but often have led to unacceptably high recurrence. Methods: We have developed a novel unilateral brace for clubfoot deformity that strategically applies patient-specific, anatomically-targeted forces to the lower limb to maintain correction. We retrospectively reviewed the cases of 26 patients with minimum follow-up of 24 months. The data were analyzed for recurrence rates, caregiver-reported compliance, and differences in Pirani score, dorsiflexion, abduction, hindfoot eversion, and resting rotation between initial and final follow-up. Results: Most patients (N = 23, 88%) were compliant with the bracing protocol. Two patients showed recurrence of deformity (8%). There were statistically significant improvements in Pirani score, dorsiflexion, abduction, hindfoot eversion, and resting external rotation. A subset of patients with sub-optimal correction at baseline showed improvement in all parameters across the course of bracing. Conclusions: This novel unilateral brace for maintenance of clubfoot correction after Ponseti treatment demonstrates rates of recurrence rates and caregiver-reported compliance at 2 years of follow up that are comparable to outcomes with traditional bilateral foot abduction orthoses.

OCT4-mediated inflammation induces cell reprogramming at the origin of cardiac valve development and calcification.

Cell plasticity plays a key role in embryos by maintaining the differentiation potential of progenitors. Whether postnatal somatic cells revert to an embryonic-like naïve state regaining plasticity and redifferentiate into a cell type leading to a disease remains intriguing. Using genetic lineage tracing and single-cell RNA sequencing, we reveal that Oct4 is induced by nuclear factor κB (NFκB) at embyronic day 9.5 in a subset of mouse endocardial cells originating from the anterior heart forming field at the onset of endocardial-to-mesenchymal transition. These cells acquired a chondro-osteogenic fate. OCT4 in adult valvular aortic cells leads to calcification of mouse and human valves. These calcifying cells originate from the Oct4 embryonic lineage. Genetic deletion of Pou5f1 (Pit-Oct-Unc, OCT4) in the endocardial cell lineage prevents aortic stenosis and calcification of ApoE−/− mouse valve. We established previously unidentified self-cell reprogramming NFκB- and OCT4-mediated inflammatory pathway triggering a dose-dependent mechanism of valve calcification.

NFκB (Nuclear Factor κ-Light-Chain Enhancer of Activated B Cells) Activity Regulates Cell-Type-Specific and Context-Specific Susceptibility to Calcification in the Aortic Valve.

OBJECTIVE: Although often studied independently, little is known about how aortic valve endothelial cells and valve interstitial cells interact collaborate to maintain tissue homeostasis or drive valve calcific pathogenesis. Inflammatory signaling is a recognized initiator of valve calcification, but the cell-type-specific downstream mechanisms have not been elucidated. In this study, we test how inflammatory signaling via NFκB (nuclear factor κ-light-chain enhancer of activated B cells) activity coordinates unique and shared mechanisms of valve endothelial cells and valve interstitial cells differentiation during calcific progression. Approach and Results: Activated NFκB was present throughout the calcific aortic valve disease (CAVD) process in both endothelial and interstitial cell populations in an established mouse model of hypercholesterolemia-induced CAVD and in human CAVD. NFκB activity induces endothelial to mesenchymal transformation in 3-dimensional cultured aortic valve endothelial cells and subsequent osteogenic calcification of transformed cells. Similarly, 3-dimensional cultured valve interstitial cells calcified via NFκB-mediated osteogenic differentiation. NFκB-mediated endothelial to mesenchymal transformation was directly demonstrated in vivo during CAVD via genetic lineage tracking. Genetic deletion of NFκB in either whole valves or valve endothelium only was sufficient to prevent valve-specific molecular and cellular mechanisms of CAVD in vivo despite the persistence of a CAVD inducing environment. CONCLUSIONS: Our results identify NFκB signaling as an essential molecular regulator for both valve endothelial and interstitial participation in CAVD pathogenesis. Direct demonstration of valve endothelial cell endothelial to mesenchymal transformation transmigration in vivo during CAVD highlights a new cellular population for further investigation in CAVD morbidity. The efficacy of valve-specific NFκB modulation in inhibiting hypercholesterolemic CAVD suggests potential benefits of multicell type integrated investigation for biological therapeutic development and evaluation for CAVD.

Notch-Tnf signalling is required for development and homeostasis of arterial valves.

AIMS: Congenital anomalies of arterial valves are common birth defects, leading to valvar stenosis. With no pharmaceutical treatment that can prevent the disease progression, prosthetic replacement is the only choice of treatment, incurring considerable morbidity and mortality. Animal models presenting localized anomalies and stenosis of congenital arterial valves similar to that of humans are critically needed research tools to uncover developmental molecular mechanisms underlying this devastating human condition. METHODS AND RESULTS: We generated and characterized mouse models with conditionally altered Notch signalling in endothelial or interstitial cells of developing valves. Mice with inactivation of Notch1 signalling in valvar endothelial cells (VEC) developed congenital anomalies of arterial valves including bicuspid aortic valves and valvar stenosis. Notch1 signalling in VEC was required for repressing proliferation and activating apoptosis of valvar interstitial cells (VIC) after endocardial-to-mesenchymal transformation (EMT). We showed that Notch signalling regulated Tnfα expression in vivo, and Tnf signalling was necessary for apoptosis of VIC and post-EMT development of arterial valves. Furthermore, activation or inhibition of Notch signalling in cultured pig aortic VEC-promoted or suppressed apoptosis of VIC, respectively. CONCLUSION: We have now met the need of critical animal models and shown that Notch-Tnf signalling balances proliferation and apoptosis for post-EMT development of arterial valves. Our results suggest that mutations in its components may lead to congenital anomaly of aortic valves and valvar stenosis in humans.

Valve interstitial cell tensional homeostasis directs calcification and extracellular matrix remodeling processes via RhoA signaling.

AIMS: Valve interstitial cells are active and aggressive players in aortic valve calcification, but their dynamic mediation of mechanically-induced calcific remodeling is not well understood. The goal of this study was to elucidate the feedback loop between valve interstitial cell and calcification mechanics using a novel three-dimensional culture system that allows investigation of the active interplay between cells, disease, and the mechanical valve environment. METHODS & RESULTS: We designed and characterized a novel bioreactor system for quantifying aortic valve interstitial cell contractility in 3-D hydrogels in control and osteogenic conditions over 14 days. Interstitial cells demonstrated a marked ability to exert contractile force on their environment and to align collagen fibers with the direction of tension. Osteogenic environment disrupted interstitial cell contractility and led to disorganization of the collagen matrix, concurrent with increased αSMA, TGF-ß, Runx2 and calcific nodule formation. Interestingly, RhoA was also increased in osteogenic condition, pointing to an aberrant hyperactivation of valve interstitial cells mechanical activity in disease. This was confirmed by inhibition of RhoA experiments. Inhibition of RhoA concurrent with osteogenic treatment reduced pro-osteogenic signaling and calcific nodule formation. Time-course correlation analysis indicated a significant correlation between interstitial cell remodeling of collagen fibers and calcification events. CONCLUSIONS: Interstitial cell contractility mediates internal stress state and organization of the aortic valve extracellular matrix. Osteogenesis disrupts interstitial cell mechanical phenotype and drives disorganization, nodule formation, and pro-calcific signaling via a RhoA-dependent mechanism.

Endothelial-derived oxidative stress drives myofibroblastic activation and calcification of the aortic valve.

AIMS: Oxidative stress is present in and contributes to calcification of the aortic valve, but the driving factors behind the initiation of valve oxidative stress are not well understood. We tested whether the valve endothelium acts as an initiator and propagator of oxidative stress in aortic valve disease. METHODS AND RESULTS: Calcified human aortic valves showed side-specific elevation of superoxide in the endothelium, co-localized with high VCAM1 expression, linking oxidative stress, inflammation, and valve degeneration. Treatment with inflammatory cytokine TNFα increased superoxide and oxidative stress and decreased eNOS and VE-cadherin acutely over 48 hours in aortic valve endothelial cells (VEC) and chronically over 21 days in ex vivo AV leaflets. Co-treatment of VEC with tetrahydrobiopterin (BH4) but not apocynin mitigated TNFα-driven VEC oxidative stress. Co-treatment of ex vivo AV leaflets with TNFα+BH4 or TNFα+peg-SOD rescued endothelial function and mitigated inflammatory responses. Both BH4 and peg-SOD rescued valve leaflets from the pro-osteogenic effects of TNFα treatment, but only peg-SOD was able to mitigate the fibrogenic effects, including increased collagen and αSMA expression. CONCLUSIONS: Aortic valve endothelial cells are a novel source of oxidative stress in aortic valve disease. TNFα-driven VEC oxidative stress causes loss of endothelial protective function, chronic inflammation, and fibrogenic and osteogenic activation, mitigated differentially by BH4 and peg-SOD. These mechanisms identify new targets for tailored antioxidant therapy focused on mitigation of oxidative stress and restoration of endothelial protection.

Heterogeneous susceptibility of valve endothelial cells to mesenchymal transformation in response to TNFα.

Lack of understanding of the early mechanisms of aortic valve stenosis and calcification hinders the development of diagnostic and therapeutic intervention strategies. Inflammation is a known component of early aortic valve disease and can induce mesenchymal transformation in a subset of aortic valve endothelial cells. Here we present a three-dimensional culture system that allows transforming and non-transforming cells to be independently isolated and analyzed. We have used the system to identify and characterize the dynamic invasion and phenotypic transition of two distinct subsets of endothelial cells: those that invade and transform under TNFα treatment, and those that resist mesenchymal transformation and remain endothelial. We determine that non-transformed cells maintain control levels of endothelial genes VE-cadherin and eNOS, while transformed cells lose these endothelial characteristics and upregulate α-smooth muscle actin. Both subsets of cells have an inflammatory phenotype marked by increased ICAM-1, but transformed cells have increased MMP-9, Notch1, TGF-ß, and BMP-4, while non-transformed cells do not. Transformed cells also have distinct effects on alignment of collagen fibers as they invade the hydrogel system, which is not found in control endothelial or interstitial valve cells. Understanding the role of transforming and non-transforming endothelial cells in valve disease will provide an important pathological link between early inflammation and later stages of disease. Discovery of the molecular signature of transformation-resistant endothelial cells could inform development of treatment strategies that promote survival of the valve endothelium.

Inflammatory cytokines promote mesenchymal transformation in embryonic and adult valve endothelial cells.

OBJECTIVE: Inflammatory activation of valve endothelium is an early phase of aortic valve disease pathogenesis, but subsequent mechanisms are poorly understood. Adult valve endothelial cells retain the developmental ability to undergo endothelial-to-mesenchymal transformation (EndMT), but a biological role has not been established. Here, we test whether and how inflammatory cytokines (tumor necrosis factor-α and interleukin-6) regulate EndMT in embryonic and adult valve endothelium. METHODS AND RESULTS: Using in vitro 3-dimensional collagen gel culture assays with primary cells, we determined that interleukin-6 and tumor necrosis factor-α induce EndMT and cell invasion in dose-dependent manners. Inflammatory-EndMT occurred through an Akt/nuclear factor-κB-dependent pathway in both adult and embryonic stages. In embryonic valves, inflammatory-EndMT required canonical transforming growth factor-ß signaling through activin receptor-like kinases 2 and 5 to drive EndMT. In adult valve endothelium, however, inflammatory-induced EndMT still occurred when activin receptor-like kinases 2 and 5 signaling was blocked. Inflammatory receptor gene expression was significantly upregulated in vivo during embryonic valve maturation. Endothelial-derived mesenchymal cells expressing activated nuclear factor-κB were found distal to calcific lesions in diseased human aortic valves. CONCLUSIONS: Inflammatory cytokine-induced EndMT in valve endothelium is present in both embryonic and adult stages, acting through Akt/nuclear factor-κB, but differently using transforming growth factor-ß signaling. Molecular signatures of valve EndMT may be important diagnostic and therapeutic targets in early valve disease.

Valvular heart diseases in the developing world: developmental biology takes center stage.

Heart valve disease is a significant and increasing global problem of which, in the developing world, the primary sufferers are the children and young adults regarded as the critical 'engine' of future economic growth. Yet, up to 10 times the current number of known sufferers remain undiagnosed in these countries. Among the most prevalent and neglected diseases are rheumatic heart disease and endomyocardial fibrosis. The etiologies of these diseases can be described in part as a dysregulation or reactivation of developmental biology pathways. Consequently, connecting mechanisms of valvulogenesis and disease etiology may represent an excellent strategy to identify therapeutic targets. These local diseases require local solutions tailored to local resources; therefore, collaboration with experienced research groups should be encouraged as a way of accelerating the creation of relevant knowledge, and its clinical translation.

The mechanobiology of mitral valve function, degeneration, and repair.

In degenerative valve disease, the highly organized mitral valve leaflet matrix stratification is progressively destroyed and replaced with proteoglycan rich, mechanically inadequate tissue. This is driven by the actions of originally quiescent valve interstitial cells that become active contractile and migratory myofibroblasts. While treatment for myxomatous mitral valve disease in humans ranges from repair to total replacement, therapies in dogs focus on treating the consequences of the resulting mitral regurgitation. The fundamental gap in our understanding is how the resident valve cells respond to altered mechanical signals to drive tissue remodeling. Despite the pathological similarities and high clinical occurrence, surprisingly little mechanistic insight has been gleaned from the dog. This review presents what is known about mitral valve mechanobiology from clinical, in vivo, and in vitro data. There are a number of experimental strategies already available to pursue this significant opportunity, but success requires the collaboration between veterinary clinicians, scientists, and engineers.

Sarcoidosis presenting as bilateral vocal cord paralysis from bilateral compression of the recurrent laryngeal nerves from thoracic adenopathy.

Bilateral true vocal fold paralysis is rarely attributable to inflammatory diseases. We describe what appears to be the first case in the medical literature of sarcoidosis presenting as isolated, bilateral true vocal cord paralysis resulting from compressive bilateral mediastinal adenopathy. The presenting symptoms, clinical outcome, radiographs and laryngeal findings are discussed in detail. Sarcoidosis should therefore be added to the differential diagnosis of bilateral vocal fold paralysis.

Spindle checkpoint protein dynamics at kinetochores in living cells.

BACKGROUND: To test current models for how unattached and untense kinetochores prevent Cdc20 activation of the anaphase-promoting complex/cyclosome (APC/C) throughout the spindle and the cytoplasm, we used GFP fusions and live-cell imaging to quantify the abundance and dynamics of spindle checkpoint proteins Mad1, Mad2, Bub1, BubR1, Mps1, and Cdc20 at kinetochores during mitosis in living PtK2 cells. RESULTS: Unattached kinetochores in prometaphase bound on average only a small fraction (estimated at 500-5000 molecules) of the total cellular pool of each spindle checkpoint protein. Measurements of fluorescence recovery after photobleaching (FRAP) showed that GFP-Cdc20 and GFP-BubR1 exhibit biphasic exponential kinetics at unattached kinetochores, with approximately 50% displaying very fast kinetics (t1/2 of approximately 1-3 s) and approximately 50% displaying slower kinetics similar to the single exponential kinetics of GFP-Mad2 and GFP-Bub3 (t1/2 of 21-23 s). The slower phase of GFP-Cdc20 likely represents complex formation with Mad2 since it was tension insensitive and, unlike the fast phase, it was absent at metaphase kinetochores that lack Mad2 but retain Cdc20 and was absent at unattached prometaphase kinetochores for the Cdc20 derivative GFP-Cdc20delta1-167, which lacks the major Mad2 binding domain but retains kinetochore localization. GFP-Mps1 exhibited single exponential kinetics at unattached kinetochores with a t1/2 of approximately 10 s, whereas most GFP-Mad1 and GFP-Bub1 were much more stable components. CONCLUSIONS: Our data support catalytic models of checkpoint activation where Mad1 and Bub1 are mainly resident, Mad2 free of Mad1, BubR1 and Bub3 free of Bub1, Cdc20, and Mps1 dynamically exchange as part of the diffuse wait-anaphase signal; and Mad2 interacts with Cdc20 at unattached kinetochores.

A prospective, randomized study of endothelin and postoperative recovery in off-pump versus conventional coronary artery bypass surgery.

OBJECTIVE: The objectives are 2-fold: (1). to serially determine endothelin (ET) levels in arterial vascular compartments in patients undergoing coronary artery bypass surgery using either cardiopulmonary bypass or off-pump techniques, and (2). to define potential relationships between endothelial levels and specific perioperative parameters of patient recovery. METHODS: In a prospective, randomized study, endothelin plasma content was measured from patients undergoing coronary artery bypass grafting using either off-pump techniques (OPCAB group, n = 25) or conventional cardiopulmonary bypass (CPB group, n = 25) before surgery, before and after coronary artery anastomosis, and 6 and 24 hours postoperatively. Specific indices of patient recovery including pulmonary artery pressures, ventilation requirement, and hospital stay were documented for patients in both study groups. RESULTS: Postoperative systemic arterial ET levels were significantly increased by 200% in the CPB group and 50% in the OPCAB group. ET levels remained significantly higher in the CPB group relative to the OPCAB group throughout the postoperative period of observation (p < 0.05). Pulmonary artery pressures, ventilation requirement, and hospital stay were significantly increased in patients in the CPB group. CONCLUSIONS: Postoperative ET levels were higher in patients who underwent CPB for coronary artery bypass surgery. Increased ET in the postoperative period may contribute to a more complex recovery from coronary artery bypass surgery in patients undergoing cardiopulmonary bypass.