Transcriptional and functional profiling identifies inflammation and endothelial-to-mesenchymal transition as potential drivers for phenotypic heterogeneity within a cohort of endothelial colony forming cells

Background: Endothelial colony-forming cells (ECFCs) derived from patients can be used to investigate pathogenic mechanisms of vascular diseases like von Willebrand disease. Considerable phenotypic heterogeneity has been observed between ECFC clones derived from healthy donors. This heterogeneity needs to be well understood in order to use ECFCs as endothelial models for disease. Objectives: Therefore, we aimed to determine phenotypic and gene expression differences between control ECFCs. Methods: A total of 34 ECFC clones derived from 16 healthy controls were analyzed. The transcriptome of a selection of ECFC clones (n = 15) was analyzed by bulk RNA sequencing and gene set enrichment analysis. Gene expression was measured in all ECFC clones by quantitative polymerase chain reaction. Phenotypic profiling was performed and migration speed of the ECFCs was measured using confocal microscopy, followed by automated quantification of cell morphometrics and migration speed. Results: Through hierarchical clustering of RNA expression profiles, we could distinguish 2 major clusters within the ECFC cohort. Major differences were associated with proliferation and migration in cluster 1 and inflammation and endothelial-to-mesenchymal transition in cluster 2. Phenotypic profiling showed significantly more and smaller ECFCs in cluster 1, which contained more and longer Weibel–Palade bodies. Migration speed in cluster 1 was also significantly higher. Conclusion: We observed a range of different RNA expression patterns between ECFC clones, mostly associated with inflammation and clear differences in Weibel–Palade body count and structure. We developed a quantitative polymerase chain reaction panel that can be used for the characterization of ECFC clones, which is essential for the correct analysis of pathogenic mechanisms in vascular disorders.</p

Characterization of large in-frame von Willebrand factor deletions highlights differing pathogenic mechanisms

Copy number variation (CNV) is known to cause all von Willebrand disease (VWD) types, although the associated pathogenic mechanisms involved have not been extensively studied. Notably, in-frame CNV provides a unique opportunity to investigate how specific von Willebrand factor (VWF) domains influence the processing and packaging of the protein. Using multiplex ligation-dependent probe amplification, this study determined the extent to which CNV contributed to VWD in the Molecular and Clinical Markers for the Diagnosis and Management of Type 1 von Willebrand Disease cohort, highlighting in-frame deletions of exons 3, 4-5, 32-34, and 33-34. Heterozygous in vitro recombinant VWF expression demonstrated that, although deletion of exons 3, 32-34, and 33-34 all resulted in significant reductions in total VWF (P < .0001, P < .001, and P < .01, respectively), only deletion of exons 3 and 32-34 had a significant impact on VWF secretion (P < .0001). High-resolution microscopy of heterozygous and homozygous deletions confirmed these observations, indicating that deletion of exons 3 and 32-34 severely impaired pseudo-Weibel-Palade body (WPB) formation, whereas deletion of exons 33-34 did not, with this variant still exhibiting pseudo-WPB formation similar to wild-type VWF. In-frame deletions in VWD, therefore, contribute to pathogenesis via moderate or severe defects in VWF biosynthesis and secretion

The development of a decision aid for patients with operable oropharyngeal carcinoma in the Netherlands - A mixed methods study

OBJECTIVE: The aim of this project is to create an interactive online patient decision aid (PDA) for oropharyngeal cancer (OPSCC) patients, eligible for transoral (robotic) surgery with an ultimate goal to assist both physicians and patients in making treatment choices. MATERIALS AND METHODS: Following the International Patient Decision Aid Standards, a mixed-methods approach was employed. The study involved semi-structured in-depth interviews with patients and physicians, thinking-out-loud sessions, and study-specific questionnaires. Thematic coding and analysis were conducted on verbatim transcriptions of audio-recorded interviews. RESULTS: The PDA drafts were evaluated by twenty OPSCC survivors and twenty multidisciplinary specialists. Significant revisions were made after phase 1 to enhance readability and reduce text, whilst incorporating videos and graphics. Following all phases, both patients and specialists rated the PDA as comprehensible, feasible, and a valuable addition to regular counseling. CONCLUSION: This study showcases the development of a PDA for early stage oropharyngeal cancer patients considering surgery and radiotherapy options. The decision aid emphasizes the disparities in short- and long-term side effects between the two treatments. Patients and physicians found the decision aid to be understandable, user-friendly, and helpful for future patients. The PDA is available on https://beslissamen.nl/

Decisional Conflict in Patients with Advanced Laryngeal Carcinoma: A Multicenter Study

Objectives: Decision-making for patients with a locally advanced laryngeal carcinoma (T3 and T4) is challenging due to the treatment choice between organ preservation and laryngectomy, both with different and high impact on function and quality of life (QoL). The complexity of these treatment decisions and their possible consequences might lead to decisional conflict (DC). This study aimed to explore the level of DC in locally advanced laryngeal carcinoma patients facing curative decision-making, and to identify possible associated factors. Methods: In this multicenter prospective cohort study, participants completed questionnaires on DC, level of shared decision-making (SDM), and a knowledge test directly after counseling and 6 months after treatment. Descriptive statistics and Spearman correlation tests were used to analyze the data. Results: Directly after counseling, almost all participants (44/45; 98%) experienced Clinically Significant DC score (CSDC >25, scale 0–100). On average, patients scored 47% (SD 20%) correct on the knowledge test. Questions related to radiotherapy were answered best (69%, SD 29%), whilst only 35% (SD 29%) of the questions related to laryngectomy were answered correctly. Patients' perceived level of SDM (scale 0–100) was 70 (mean, SD 16.2), and for physicians this was 70 (SD 1.7). Conclusion: Most patients with advanced larynx cancer experience high levels of DC. Low knowledge levels regarding treatment aspects indicate a need for better patient counseling. Level of Evidence: 4 Laryngoscope, 134:3604–3610, 2024

Automated segmentation and quantitative analysis of organelle morphology, localization and content using CellProfiler.

One of the most used and versatile methods to study number, dimensions, content and localization of secretory organelles is confocal microscopy analysis. However, considerable heterogeneity exists in the number, size and shape of secretory organelles that can be present in the cell. One thus needs to analyze large numbers of organelles for valid quantification. Properly evaluating these parameters requires an automated, unbiased method to process and quantitatively analyze microscopy data. Here, we describe two pipelines, run by CellProfiler software, called OrganelleProfiler and OrganelleContentProfiler. These pipelines were used on confocal images of endothelial colony forming cells (ECFCs), which contain unique secretory organelles called Weibel-Palade bodies (WPBs), and on early endosomes in ECFCs and human embryonic kidney 293T (HEK293T) cells. Results show that the pipelines can quantify the cell count, size, organelle count, organelle size, shape, relation to cells and nuclei, and distance to these objects in both endothelial and HEK293T cells. Additionally, the pipelines were used to measure the reduction in WPB size after disruption of the Golgi and to quantify the perinuclear clustering of WPBs after triggering of cAMP-mediated signaling pathways in ECFCs. Furthermore, the pipeline is able to quantify secondary signals located in or on the organelle or in the cytoplasm, such as the small WPB GTPase Rab27A. Cell profiler measurements were checked for validity using Fiji. To conclude, these pipelines provide a powerful, high-processing quantitative tool for the characterization of multiple cell and organelle types. These pipelines are freely available and easily editable for use on different cell types or organelles

3D stereophotogrammetry for the assessment of tracheostoma anatomy

Conclusion. 3D stereophotogrammetry is a useful tool for quantitative assessment of tracheostoma anatomy, and thus in future could possibly play a role in solving current problems with peristomal and intratracheal fixation of stoma appliances. Objectives. Differences in tracheostoma anatomy between laryngectomized patients are held partly responsible for the differences in duration of attachment of standard peristomal adhesives for heat and moisture exchangers (HMEs) and automatic stoma valves (ASVs). Accurate information on a set of basic stoma anatomy parameters should be helpful to overcome fixation problems in individual patients and in the general laryngectomy population. Patients and methods. This was an exploratory observational pilot study in 20 laryngectomized patients. Three-dimensional (3D) images of the tracheostoma and surrounding tissue were captured with a 3D digital camera, which applies the advanced principles of stereophotogrammetry. Data were analysed by using a 3D editing program. The following tracheostoma parameters were measured for each patient: horizontal and vertical diameters, circumference, depth and surface. Results. Inter-observer outcomes show a mean stoma circumference of 58.2 mm (SD 11.6 mm). The mean surface was calculated at 186.6 mm2 (SD 61 mm(2)). The mean size of the stoma's horizontal diameter and vertical diameter was 14.7 mm (SD 2.9 mm) and 19.0 mm (SD 3.4 mm), respectively. The mean depth of the stoma in relation to the right sternal head of the m. sternocleidomastoideus was 15.5 mm (SD 5.4 mm) and in relation to the left it was 16 mm (SD 5.6 mm)

Storage and secretion of naturally occurring von Willebrand factor A domain variants

Von Willebrand disease (VWD) is a bleeding disorder characterized by reduced plasma von Willebrand factor (VWF) levels or functionally abnormal VWF. Low VWF plasma levels in VWD patients are the result of mutations in the VWF gene that lead to decreased synthesis, impaired secretion, increased clearance or a combination thereof. However, expression studies of variants located in the A domains of VWF are limited. We therefore characterized the biosynthesis of VWF mutations, located in the VWF A1-A3 domains, that were found in families diagnosed with VWD. Human Embryonic Kidney 293 (HEK293) cells were transiently transfected with plasmids encoding full-length wild-type VWF or mutant VWF. Six mutations in the A1-A3 domains were expressed. We found that all mutants, except one, showed impaired formation of elongated pseudo-Weibel-Palade bodies (WPB). In addition, two mutations also showed reduced numbers of pseudo-WPB, even in the heterozygous state, and increased endoplasmic reticulum retention, which is in accordance with the impaired regulated secretion seen in patients. Regulated secretion upon stimulation of transfected cells reproduced the in vivo situation, indicating that HEK293 cells expressing VWF variants found in patients with VWD can be used to properly assess defects in regulated secretion

Biogenesis of Weibel-Palade bodies in von Willebrand’s disease variants with impaired von Willebrand factor intrachain or interchain disulfide bond formation

Background Mutations of cysteine residues in von Willebrand factor are known to reduce the storage and secretion of this factor, thus leading to reduced antigen levels. However, one cysteine mutation, p.Cys2773Ser, has been found in patients with type 2A(IID) von Willebrand's disease who have normal plasma levels of von Willebrand factor. We hypothesize that disruption of either intra-or interchain disulfide bonds by cysteine mutations in von Willebrand factor has different effects on the biogenesis of Weibel-Palade bodies. Design and Methods The effect of specific cysteine mutations that either disrupt intrachain (p.Cys1130Phe and p.Cys2671Tyr) or interchain (p.Cys2773Ser) disulfide bonds on storage and secretion of von Willebrand factor was studied by transient transfection of human embryonic kidney cell line 293. Upon expression of von Willebrand factor these cells formed endothelial Weibel-Palade body-like organelles called pseudo-Weibel-Palade bodies. Storage of von Willebrand factor was analyzed with both confocal immunofluorescence and electron microscopy. Regulated secretion of von Willebrand factor was induced by phorbol 12-myristate 13-acetate. Results p.Cys1130Phe and p.Cys2671Tyr reduced the storage of von Willebrand factor into pseudo-Weibel-Palade bodies with notable retention of von Willebrand factor in the endoplasmic reticulum, whereas p.Cys2773Ser-von Willebrand factor was stored normally. As expected, wild-type von Willebrand factor formed proteinaceous tubules that were seen under electron microscopy as longitudinal striations in pseudo-Weibel-Palade bodies. p.Cys2773Ser caused severe defects in von Willebrand factor multimerization but the factor formed normal tubules. Furthermore, the basal and regulated secretion of von Willebrand factor was drastically impaired by p.Cys1130Phe and p.Cys2671Tyr, but not by p.Cys2773Ser. Conclusions We postulate that natural mutations of cysteines involved in the formation of interchain disulfide bonds do not affect either the storage in Weibel-Palade bodies or secretion of von Willebrand factor, whereas mutations of cysteines forming intrachain disulfide bonds lead to reduced von Willebrand factor storage and secretion because the von Willebrand factor is retained in the endoplasmic reticulu