Emerging Trends in Left Ventricular Thrombus: A Comprehensive Review of Non-Ischemic and Ischemic Cardiopathies, Including Eosinophilic Myocarditis, Chagas Cardiomyopathy, Amyloidosis, and Innovative Anticoagulant Approaches.

This comprehensive review explores the intricate aspects of left ventricular thrombus (LVT), a potential complication in both ischemic and non-ischemic cardiomyopathies. It provides a thorough understanding of left ventricular thrombus, revealing its uncommon incidence in the general population (7 cases per 10,000 patients), predominantly linked to ischemic heart diseases (ICMs) at an 80% prevalence rate. Diagnostic tools, notably transthoracic echocardiography (TTE) and cardiac magnetic resonance imaging (CMR), demonstrate varying sensitivity but remain indispensable in specific clinical contexts related to LVT as non-invasive diagnostic modalities. A detailed comparison between ICM patients and those with non-ischemic cardiomyopathy (NICM) who have left ventricular thrombus reveals subtle distinctions with significant clinical implications. This analysis underscores the importance of these imaging techniques in distinguishing between the two conditions. Additionally, we explored the occurrence of LVT in specific non-ischemic cardiomyopathies, including Takotsubo syndrome, hypertrophic cardiomyopathy, eosinophilic myocarditis, Chagas disease, cardiac amyloidosis, and several other conditions. The article further delves into anticoagulation strategies, thoroughly examining their impact on LVT regression and patient outcomes. Pharmacological interventions, with a focus on direct oral anticoagulants, emerge as promising alternatives; however, there is insufficient information on their efficiency and safety, especially in NICM population. In conclusion, this review highlights the complex nature of LVT, incorporating a range of etiopathogenic factors, diagnostic complexities, and evolving therapeutic approaches. It emphasizes the pressing need for ongoing research in this field.

Unmasking Pandemic Echoes: An In-Depth Review of Long COVID's Unabated Cardiovascular Consequences beyond 2020.

At the beginning of 2020, coronavirus disease 2019 (COVID-19) emerged as a new pandemic, leading to a worldwide health crisis and overwhelming healthcare systems due to high numbers of hospital admissions, insufficient resources, and a lack of standardized therapeutic protocols. Multiple genetic variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been detected since its first public declaration in 2020, some of them being considered variants of concern (VOCs) corresponding to several pandemic waves. Nevertheless, a growing number of COVID-19 patients are continuously discharged from hospitals, remaining symptomatic even months after their first episode of COVID-19 infection. Long COVID-19 or 'post-acute COVID-19 syndrome' emerged as the new pandemic, being characterized by a high variability of clinical manifestations ranging from cardiorespiratory and neurological symptoms such as chest pain, exertional dyspnoea or cognitive disturbance to psychological disturbances, e.g., depression, anxiety or sleep disturbance with a crucial impact on patients' quality of life. Moreover, Long COVID is viewed as a new cardiovascular risk factor capable of modifying the trajectory of current and future cardiovascular diseases, altering the patients' prognosis. Therefore, in this review we address the current definitions of Long COVID and its pathophysiology, with a focus on cardiovascular manifestations. Furthermore, we aim to review the mechanisms of acute and chronic cardiac injury and the variety of cardiovascular sequelae observed in recovered COVID-19 patients, in addition to the potential role of Long COVID clinics in the medical management of this new condition. We will further address the role of future research for a better understanding of the actual impact of Long COVID and future therapeutic directions.

An imaging artifact unmasked by contrast-enhanced transesophageal echocardiography.

Performing transesophageal imaging of the left atrial appendage is key before cardioversion for atrial fibrillation. Ultrasound artifacts may induce misinterpretation and decrease in confidence for thrombus exclusion.

A conformational transition of the D'D3 domain primes von Willebrand factor for multimerization.

Von Willebrand factor (VWF) is a multimeric plasma glycoprotein that is critically involved in hemostasis. Biosynthesis of long VWF concatemers in the endoplasmic reticulum and the trans-Golgi is still not fully understood. We use the single-molecule force spectroscopy technique magnetic tweezers to analyze a previously hypothesized conformational change in the D'D3 domain crucial for VWF multimerization. We find that the interface formed by submodules C8-3, TIL3, and E3 wrapping around VWD3 can open and expose 2 buried cysteines, Cys1099 and Cys1142, that are vital for multimerization. By characterizing the conformational change at varying levels of force, we can quantify the kinetics of the transition and stability of the interface. We find a pronounced destabilization of the interface on lowering the pH from 7.4 to 6.2 and 5.5. This is consistent with initiation of the conformational change that enables VWF multimerization at the D'D3 domain by a decrease in pH in the trans-Golgi network and Weibel-Palade bodies. Furthermore, we find a stabilization of the interface in the presence of coagulation factor VIII, providing evidence for a previously hypothesized binding site in submodule C8-3. Our findings highlight the critical role of the D'D3 domain in VWF biosynthesis and function, and we anticipate our methodology to be applicable to study other, similar conformational changes in VWF and beyond.

Endotoxinemia Accelerates Atherosclerosis Through Electrostatic Charge-Mediated Monocyte Adhesion.

BACKGROUND: Acute infection is a well-established risk factor of cardiovascular inflammation increasing the risk for a cardiovascular complication within the first weeks after infection. However, the nature of the processes underlying such aggravation remains unclear. Lipopolysaccharide derived from Gram-negative bacteria is a potent activator of circulating immune cells including neutrophils, which foster inflammation through discharge of neutrophil extracellular traps (NETs). Here, we use a model of endotoxinemia to link acute infection and subsequent neutrophil activation with acceleration of vascular inflammation Methods: Acute infection was mimicked by injection of a single dose of lipopolysaccharide into hypercholesterolemic mice. Atherosclerosis burden was studied by histomorphometric analysis of the aortic root. Arterial myeloid cell adhesion was quantified by intravital microscopy. RESULTS: Lipopolysaccharide treatment rapidly enhanced atherosclerotic lesion size by expansion of the lesional myeloid cell accumulation. Lipopolysaccharide treatment led to the deposition of NETs along the arterial lumen, and inhibition of NET release annulled lesion expansion during endotoxinemia, thus suggesting that NETs regulate myeloid cell recruitment. To study the mechanism of monocyte adhesion to NETs, we used in vitro adhesion assays and biophysical approaches. In these experiments, NET-resident histone H2a attracted monocytes in a receptor-independent, surface charge-dependent fashion. Therapeutic neutralization of histone H2a by antibodies or by in silico designed cyclic peptides enables us to reduce luminal monocyte adhesion and lesion expansion during endotoxinemia. CONCLUSIONS: Our study shows that NET-associated histone H2a mediates charge-dependent monocyte adhesion to NETs and accelerates atherosclerosis during endotoxinemia.

Designed anchoring geometries determine lifetimes of biotin-streptavidin bonds under constant load and enable ultra-stable coupling.

The small molecule biotin and the homotetrameric protein streptavidin (SA) form a stable and robust complex that plays a pivotal role in many biotechnological and medical applications. In particular, the SA-biotin linkage is frequently used in single-molecule force spectroscopy (SMFS) experiments. Recent data suggest that SA-biotin bonds show strong directional dependence and a broad range of multi-exponential lifetimes under load. Here, we investigate engineered SA variants with different valencies and a unique tethering point under constant forces using a magnetic tweezers assay. We observed orders-of-magnitude differences in the lifetimes under force, which we attribute to the distinct force-loading geometries in the different SA variants. Lifetimes showed exponential dependencies on force, with extrapolated lifetimes at zero force that are similar for the different SA variants and agree with parameters determined from constant-speed dynamic SMFS experiments. We identified an especially long-lived tethering geometry that will facilitate ultra-stable SMFS experiments.

Inadequate tissue mineralization promotes cancer cell attachment.

Bone metastases are a frequent complication in prostate cancer, and several studies have shown that vitamin D deficiency promotes bone metastases. However, while many studies focus on vitamin D's role in cell metabolism, the effect of chronically low vitamin D levels on bone tissue, i.e. insufficient mineralization of the tissue, has largely been ignored. To investigate, whether poor tissue mineralization promotes cancer cell attachment, we used a fluorescence based adhesion assay and single cell force spectroscopy to quantify the adhesion of two prostate cancer cell lines to well-mineralized and demineralized dentin, serving as biomimetic bone model system. Adhesion rates of bone metastases-derived PC3 cells increased significantly on demineralized dentin. Additionally, on mineralized dentin, PC3 cells adhered mainly via membrane anchored surface receptors, while on demineralized dentin, they adhered via cytoskeleton-anchored transmembrane receptors, pointing to an interaction via exposed collagen fibrils. The adhesion rate of lymph node derived LNCaP cells on the other hand is significantly lower than that of PC3 and not predominately mediated by cytoskeleton-linked receptors. This indicates that poor tissue mineralization facilitates the adhesion of invasive cancer cells by the exposure of collagen and emphasizes the disease modifying effect of sufficient vitamin D for cancer patients.

Multiplexed protein force spectroscopy reveals equilibrium protein folding dynamics and the low-force response of von Willebrand factor.

Single-molecule force spectroscopy has provided unprecedented insights into protein folding, force regulation, and function. So far, the field has relied primarily on atomic force microscope and optical tweezers assays that, while powerful, are limited in force resolution, throughput, and require feedback for constant force measurements. Here, we present a modular approach based on magnetic tweezers (MT) for highly multiplexed protein force spectroscopy. Our approach uses elastin-like polypeptide linkers for the specific attachment of proteins, requiring only short peptide tags on the protein of interest. The assay extends protein force spectroscopy into the low force (<1 pN) regime and enables parallel and ultra-stable measurements at constant forces. We present unfolding and refolding data for the small, single-domain protein ddFLN4, commonly used as a molecular fingerprint in force spectroscopy, and for the large, multidomain dimeric protein von Willebrand factor (VWF) that is critically involved in primary hemostasis. For both proteins, our measurements reveal exponential force dependencies of unfolding and refolding rates. We directly resolve the stabilization of the VWF A2 domain by Ca2+ and discover transitions in the VWF C domain stem at low forces that likely constitute the first steps of VWF's mechano-activation. Probing the force-dependent lifetime of biotin-streptavidin bonds, we find that monovalent streptavidin constructs with specific attachment geometry are significantly more force stable than commercial, multivalent streptavidin. We expect our modular approach to enable multiplexed force-spectroscopy measurements for a wide range of proteins, in particular in the physiologically relevant low-force regime.

Advancing multimer analysis of von Willebrand factor by single-molecule AFM imaging.

The formation of hemostatic plugs at sites of vascular injury crucially involves the multimeric glycoprotein von Willebrand factor (VWF). VWF multimers are linear chains of N-terminally linked dimers. The latter are formed from monomers via formation of the C-terminal disulfide bonds Cys2771-Cys2773', Cys2773-Cys2771', and Cys2811-Cys2811'. Mutations in VWF that impair multimerization can lead to subtype 2A of the bleeding disorder von Willebrand Disease (VWD). Commonly, the multimer size distribution of VWF is assessed by electrophoretic multimer analysis. Here, we present atomic force microscopy (AFM) imaging as a method to determine the size distribution of VWF variants by direct visualization at the single-molecule level. We first validated our approach by investigating recombinant wildtype VWF and a previously studied mutant (p.Cys1099Tyr) that impairs N-terminal multimerization. We obtained excellent quantitative agreement with results from earlier studies and with electrophoretic multimer analysis. We then imaged specific mutants that are known to exhibit disturbed C-terminal dimerization. For the mutants p.Cys2771Arg and p.Cys2773Arg, we found the majority of monomers (87 ± 5% and 73 ± 4%, respectively) not to be C-terminally dimerized. While these results confirm that Cys2771 and Cys2773 are crucial for dimerization, they additionally provide quantitative information on the mutants' different abilities to form alternative C-terminal disulfides for residual dimerization. We further mutated Cys2811 to Ala and found that only 23 ± 3% of monomers are not C-terminally dimerized, indicating that Cys2811 is structurally less important for dimerization. Furthermore, for mutants p.Cys2771Arg, p.Cys2773Arg, and p.Cys2811Ala we found 'even-numbered' non-native multimers, i.e. multimers with monomers attached on both termini; a multimer species that cannot be distinguished from native multimers by conventional multimer analysis. Summarizing, we demonstrate that AFM imaging can provide unique insights into VWF processing defects at the single-molecule level that cannot be gained from established methods of multimer analysis.

Heath risk among pesticide sellers in Bamenda (Cameroon) and peripheral areas.

In Bamenda and peripheral zones, studies have been focused on the effects of pesticides on farmers (pesticide users) while nothing has been done to assess the exposure of sellers to pesticides. This study aimed at evaluating the exposure of pesticide sellers in the same area. Thirty-two questionnaires were administered to 32 pesticide sellers systematically selected, and chi-square was used for statistical analysis. From each shop, a respondent was chosen among the workers according to its daily time spent in the workplace. The results showed that there is similarity between sellers in Bamenda and peripheral area; one active ingredient (metalaxyl) and one formulation (beauchamp) sold are not registered; throat irritation, headaches, fatigue, skin irritation, eye irritation, and difficulty in breathing with more cases of nose irritation were symptoms observed; pesticides are stored either in the shops or in warehouses; safety measures generally applied are sitting outside the shop, taking medicated charcoal and the use of protective clothing; 56% have less than 5 years experience. Permanent pesticide sellers are then exposed to chronic intoxication in Bamenda and neighboring zones. Employers should make use of protective clothing in their shops when manipulating pesticides in the application of safety measures.

Biophysical approaches promote advances in the understanding of von Willebrand factor processing and function.

The large multimeric plasma glycoprotein von Willebrand factor (VWF) is essential for primary hemostasis by recruiting platelets to sites of vascular injury. VWF multimers respond to elevated hydrodynamic forces by elongation, thereby increasing their adhesiveness to platelets. Thus, the activation of VWF is force-induced, as is its inactivation. Due to these attributes, VWF is a highly interesting system from a biophysical point of view, and is well suited for investigation using biophysical approaches. Here, we give an overview on recent studies that predominantly employed biophysical methods to gain novel insights into multiple aspects of VWF: Electron microscopy was used to shed light on the domain structure of VWF and the mechanism of VWF secretion. High-resolution stochastic optical reconstruction microscopy, atomic force microscopy (AFM), microscale thermophoresis and fluorescence correlation spectroscopy allowed identification of protein disulfide isomerase isoform A1 as the VWF dimerizing enzyme and, together with molecular dynamics simulations, postulation of the dimerization mechanism. Advanced mass spectrometry led to detailed identification of the glycan structures carried by VWF. Microfluidics was used to illustrate the interplay of force and VWF function. Results from optical tweezers measurements explained mechanisms of the force-dependent functions of VWF's domains A1 and A2 and, together with thermodynamic approaches, increased our understanding of mutation-induced dysfunctions of platelet-binding. AFM-based force measurements and AFM imaging enabled exploration of intermonomer interactions and their dependence on pH and divalent cations. These advances would not have been possible by the use of biochemical methods alone and show the benefit of interdisciplinary research approaches.

pH-Dependent Interactions in Dimers Govern the Mechanics and Structure of von Willebrand Factor.

Von Willebrand factor (VWF) is a multimeric plasma glycoprotein that is activated for hemostasis by increased hydrodynamic forces at sites of vascular injury. Here, we present data from atomic force microscopy-based single-molecule force measurements, atomic force microscopy imaging, and small-angle x-ray scattering to show that the structure and mechanics of VWF are governed by multiple pH-dependent interactions with opposite trends within dimeric subunits. In particular, the recently discovered strong intermonomer interaction, which induces a firmly closed conformation of dimers and crucially involves the D4 domain, was observed with highest frequency at pH 7.4, but was essentially absent at pH values below 6.8. However, below pH 6.8, the ratio of compact dimers increased with decreasing pH, in line with a previous transmission electron microscopy study. These findings indicated that the compactness of dimers at pH values below 6.8 is promoted by other interactions that possess low mechanical resistance compared with the strong intermonomer interaction. By investigating deletion constructs, we found that compactness under acidic conditions is primarily mediated by the D4 domain, i.e., remarkably by the same domain that also mediates the strong intermonomer interaction. As our data suggest that VWF has the highest mechanical resistance at physiological pH, local deviations from physiological pH (e.g., at sites of vascular injury) may represent a means to enhance VWF's hemostatic activity where needed.

Force sensing by the vascular protein von Willebrand factor is tuned by a strong intermonomer interaction.

The large plasma glycoprotein von Willebrand factor (VWF) senses hydrodynamic forces in the bloodstream and responds to elevated forces with abrupt elongation, thereby increasing its adhesiveness to platelets and collagen. Remarkably, forces on VWF are elevated at sites of vascular injury, where VWF's hemostatic potential is important to mediate platelet aggregation and to recruit platelets to the subendothelial layer. Adversely, elevated forces in stenosed vessels lead to an increased risk of VWF-mediated thrombosis. To dissect the remarkable force-sensing ability of VWF, we have performed atomic force microscopy (AFM)-based single-molecule force measurements on dimers, the smallest repeating subunits of VWF multimers. We have identified a strong intermonomer interaction that involves the D4 domain and critically depends on the presence of divalent ions, consistent with results from small-angle X-ray scattering (SAXS). Dissociation of this strong interaction occurred at forces above [Formula: see text]50 pN and provided [Formula: see text]80 nm of additional length to the elongation of dimers. Corroborated by the static conformation of VWF, visualized by AFM imaging, we estimate that in VWF multimers approximately one-half of the constituent dimers are firmly closed via the strong intermonomer interaction. As firmly closed dimers markedly shorten VWF's effective length contributing to force sensing, they can be expected to tune VWF's sensitivity to hydrodynamic flow in the blood and to thereby significantly affect VWF's function in hemostasis and thrombosis.

Decoding Cytoskeleton-Anchored and Non-Anchored Receptors from Single-Cell Adhesion Force Data.

Complementary to parameters established for cell-adhesion force curve analysis, we evaluated the slope before a force step together with the distance from the surface at which the step occurs and visualized the result in a two-dimensional density plot. This new tool allows detachment steps of long membrane tethers to be distinguished from shorter jumplike force steps, which are typical for cytoskeleton-anchored bonds. A prostate cancer cell line (PC3) immobilized on an atomic-force-microscopy sensor interacted with three different substrates: collagen-I (Col-I), bovine serum albumin, and a monolayer of bone marrow-derived stem cells (SCP1). To address PC3 cells' predominant Col-I binding molecules, an antibody-blocking ß1-integrin was used. Untreated PC3 cells on Col-I or SCP1 cells, which express Col-I, predominantly showed jumps in their force curves, while PC3 cells on bovine-serum-albumin- and antibody-treated PC3 cells showed long membrane tethers. The probability density plots thus revealed that ß1-integrin-specific interactions are predominately anchored to the cytoskeleton, while the nonspecific interactions are mainly membrane-anchored. Experiments with latrunculin-A-treated PC3 cells corroborated these observations. The plots thus reveal details of the anchoring of bonds to the cell and provide a better understanding of receptor-ligand interactions.

A fast recoiling silk-like elastomer facilitates nanosecond nematocyst discharge.

BACKGROUND: The discharge of the Cnidarian stinging organelle, the nematocyst, is one of the fastest processes in biology and involves volume changes of the highly pressurised (150 bar) capsule of up to 50%. Hitherto, the molecular basis for the unusual biomechanical properties of nematocysts has been elusive, as their structure was mainly defined as a stress-resistant collagenous matrix. RESULTS: Here, we characterise Cnidoin, a novel elastic protein identified as a structural component of Hydra nematocysts. Cnidoin is expressed in nematocytes of all types and immunostainings revealed incorporation into capsule walls and tubules concomitant with minicollagens. Similar to spider silk proteins, to which it is related at sequence level, Cnidoin possesses high elasticity and fast coiling propensity as predicted by molecular dynamics simulations and quantified by force spectroscopy. Recombinant Cnidoin showed a high tendency for spontaneous aggregation to bundles of fibrillar structures. CONCLUSIONS: Cnidoin represents the molecular factor involved in kinetic energy storage and release during the ultra-fast nematocyst discharge. Furthermore, it implies an early evolutionary origin of protein elastomers in basal metazoans.

Exponential size distribution of von Willebrand factor.

Von Willebrand Factor (VWF) is a multimeric protein crucial for hemostasis. Under shear flow, it acts as a mechanosensor responding with a size-dependent globule-stretch transition to increasing shear rates. Here, we quantify for the first time, to our knowledge, the size distribution of recombinant VWF and VWF-eGFP using a multilateral approach that involves quantitative gel analysis, fluorescence correlation spectroscopy, and total internal reflection fluorescence microscopy. We find an exponentially decaying size distribution of multimers for recombinant VWF as well as for VWF derived from blood samples in accordance with the notion of a step-growth polymerization process during VWF biosynthesis. The distribution is solely described by the extent of polymerization, which was found to be reduced in the case of the pathologically relevant mutant VWF-IIC. The VWF-specific protease ADAMTS13 systematically shifts the VWF size distribution toward smaller sizes. This dynamic evolution is monitored using fluorescence correlation spectroscopy and compared to a computer simulation of a random cleavage process relating ADAMTS13 concentration to the degree of VWF breakdown. Quantitative assessment of VWF size distribution in terms of an exponential might prove to be useful both as a valuable biophysical characterization and as a possible disease indicator for clinical applications.

Increase in lens capsule stiffness caused by vital dyes.

PURPOSE: To assess potential changes in lens capsule mechanical properties after staining with brilliant blue, indocyanine green (ICG), and trypan blue. SETTING: Department of Ophthalmology and Applied Physics and Center for NanoScience, Ludwig-Maximilians-University, Munich, Germany. DESIGN: Experimental study. METHODS: Fifteen unstained lens capsules were dissected into 7 wedge-shaped parts. Three fragments were stained with brilliant blue 0.025%, ICG 0.05%, and trypan blue 0.06%, respectively, for 1 minute. Another 3 specimens were additionally illuminated using a standard light source. The seventh part served as an untreated control. All specimens were analyzed using atomic force microscopy (AFM) in contact mode with a scan rate of 0.6 Hz. Two scan regions of 10 µm × 10 µm were chosen, and stiffness was determined using AFM in a force spectroscopy mode. The force curves were performed with a data rate of 5000 Hz. RESULTS: Staining of the samples resulted in an increase in tissue stiffness (brilliant blue: P<.001; ICG: P<.01; trypan blue: P<.05). Additional illumination after staining further increased tissue stiffness, but not significantly. Mean increase in the relative elasticity values were 1.61 ± 0.15 (SD) for brilliant blue, 2.04 ± 0.21 for brilliant blue with illumination, 1.63 ± 0.22 for ICG, 2.01 ± 0.22 for ICG with illumination, 1.23 ± 0.11 for trypan blue, and 1.39 ± 0.11 for trypan blue with illumination. In relation to unstained tissue, the relative elasticity of the stained tissue increased 1.2-fold after illumination. CONCLUSION: Staining significantly increased the mechanical properties of the human lens capsule. FINANCIAL DISCLOSURE: No author has a financial or proprietary interest in any material or method mentioned.

Probing the interaction forces of prostate cancer cells with collagen I and bone marrow derived stem cells on the single cell level.

Adhesion of metastasizing prostate carcinoma cells was quantified for two carcinoma model cell lines LNCaP (lymph node-specific) and PC3 (bone marrow-specific). By time-lapse microscopy and force spectroscopy we found PC3 cells to preferentially adhere to bone marrow-derived mesenchymal stem cells (SCP1 cell line). Using atomic force microscopy (AFM) based force spectroscopy, the mechanical pattern of the adhesion to SCP1 cells was characterized for both prostate cancer cell lines and compared to a substrate consisting of pure collagen type I. PC3 cells dissipated more energy (27.6 aJ) during the forced de-adhesion AFM experiments and showed significantly more adhesive and stronger bonds compared to LNCaP cells (20.1 aJ). The characteristic signatures of the detachment force traces revealed that, in contrast to the LNCaP cells, PC3 cells seem to utilize their filopodia in addition to establish adhesive bonds. Taken together, our study clearly demonstrates that PC3 cells have a superior adhesive affinity to bone marrow mesenchymal stem cells, compared to LNCaP. Semi-quantitative PCR on both prostate carcinoma cell lines revealed the expression of two Col-I binding integrin receptors, α1ß1 and α2ß1 in PC3 cells, suggesting their possible involvement in the specific interaction to the substrates. Further understanding of the exact mechanisms behind this phenomenon might lead to optimized therapeutic applications targeting the metastatic behavior of certain prostate cancer cells towards bone tissue.