No abstract

Summary Von Willebrand disease (VWD) is the most common inherited bleeding disorder but its diagnosis can be challenging due to the heterogeneity of the disease. VWD is mainly associated with mild mucocutaneous bleeding, although there are more severe phenotypes with bleeding from the gastrointestinal tract or even the joints. Also, surgical interventions and trauma may lead to critical bleeding events. These bleeding episodes are all related to quantitative or qualitative defects of von Willebrand factor (VWF), a multimeric glycoprotein produced by endothelial cells and megakaryocytes, which mediates platelet adhesion and aggregation and binds factor VIII (FVIII) in the circulation. This review describes the diagnostic procedures required for correct diagnosis. Accurate diagnosis and classification is required for proper treatment and counseling. Assessment of bleeding starts with the medical history. After a positive bleeding or family history, subsequent laboratory investigations will start with a panel of standard screening tests for hemostatic defects. Patients suspected of having VWD will be tested for plasma VWF antigen levels, the ability of VWF to bind platelets and FVIII activity. When VWD is confirmed, a set of subtyping tests can classify the patients as VWD types 1, 2 (A, B, M or N) or 3. The performance of some additional assays and analyses, such as VWF propeptide measurement or genetic analysis, may help in identifying the pathological mechanism behind certain defects or can guide in the choice of treatment.

Facioscapulohumeral muscular dystrophy is caused by incomplete epigenetic repression of the transcription factor DUX4 in skeletal muscle. A copy of DUX4 is located within each unit of the D4Z4 macrosatellite repeat array and its derepression in somatic cells is caused by either repeat array contraction (FSHD1) or by mutations in the chromatin repressor SMCHD1 (FSHD2). While DUX4 expression has thus far only been detected in FSHD muscle and muscle cell cultures, and increases with in vitro myogenic differentiation, the D4Z4 chromatin structure has only been studied in proliferating myoblasts or non-myogenic cells. We here show that SMCHD1 protein levels at D4Z4 decline during muscle cell differentiation and correlate with DUX4 derepression. In FSHD2, but not FSHD1, the loss of SMCHD1 repressor activity is partially compensated by increased Polycomb Repressive Complex 2 (PRC2)–mediated H3K27 trimethylation at D4Z4, a situation that can be mimicked by SMCHD1 knockdown in control myotubes. In contrast, moderate overexpression of SMCHD1 results in DUX4 silencing in FSHD1 and FSHD2 myotubes demonstrating that DUX4 derepression in FSHD is reversible. Together, we show that in FSHD1 and FSHD2 the decline in SMCHD1 protein levels during muscle cell differentiation renders skeletal muscle sensitive to DUX4.

SUMMARY Psychological stress (PS) is associated with systemic inflammation and accelerates inflammatory disease progression (e.g., atherosclerosis). The mechanisms underlying stress-mediated inflammation and future health risk are poorly understood. Monocytes are key in sustaining systemic inflammation, and recent studies demonstrate that they maintain the memory of inflammatory insults, leading to a heightened inflammatory response upon rechallenge. We show that PS induces remodeling of the chromatin landscape and transcriptomic reprogramming of monocytes, skewing them to a primed hyperinflammatory phenotype. Monocytes from stressed mice and humans exhibit a characteristic inflammatory transcriptomic signature and are hyperresponsive upon stimulation with Toll-like receptor ligands. RNA and ATAC sequencing reveal that monocytes from stressed mice and humans exhibit activation of metabolic pathways (mTOR and PI3K) and reduced chromatin accessibility at mitochondrial respiration-associated loci. Collectively, our findings suggest that PS primes the reprogramming of myeloid cells to a hyperresponsive inflammatory state, which may explain how PS confers inflammatory disease risk.

Background Treatment of the bleeding disorder von Willebrand disease (VWD) focuses on increasing von Willebrand factor (VWF) levels by administration of desmopressin or VWF-containing concentrates. Both therapies leave the production of mutant VWF unhindered, which may have additional consequences, such as thrombocytopenia in patients with VWD type 2B, competition between mutant and normal VWF for platelet receptors, and the potential development of intestinal angiodysplasia. Most cases of VWD are caused by dominant-negative mutations in VWF, and we hypothesize that diminishing expression of mutant VWF positively affects VWD phenotypes. Objectives To investigate allele-specific inhibition of VWF by applying small interfering RNAs (siRNAs) targeting common single-nucleotide polymorphisms (SNPs) in VWF. This approach allows allele-specific knockdown irrespective of the mutations causing VWD. Methods Four SNPs with a high predicted heterozygosity within VWF were selected, and siRNAs were designed against both alleles of the four SNPs. siRNA efficiency, allele specificity and siRNA-mediated phenotypic improvements were determined in VWF-expressing HEK293 cells. Results Twelve siRNAs were able to efficiently inhibit single VWF alleles in HEK293 cells that stably produce VWF. Transient cotransfections of these siRNAs with two VWF alleles resulted in a clear preference for the targeted allele over the untargeted allele for 11 siRNAs. We also demonstrated siRNA-mediated phenotypic improvement of the VWF multimerization pattern of the VWD type 2A mutation VWF p.Cys2773Ser. Conclusions Allele-specific siRNAs are able to distinguish VWF alleles on the basis of one nucleotide variation, and are able to improve a severe multimerization defect caused by VWF p.Cys2773Ser. This holds promise for the therapeutic application of allele-specific siRNAs in dominant-negative VWD.

This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

Essentials Endothelial colony forming cells (ECFCs) are a powerful tool to study vascular diseases ex vivo.Separate ECFC lines show variations in morphology and von Willebrand factor‐related parameters.Maximum cell density is correlated with von Willebrand factor expression in ECFCs.Variations in ECFC lines are dependent on the age and mesenchymal state of the cells. AbstractBackgroundEndothelial colony forming cells (ECFCs) are cultured endothelial cells derived from peripheral blood. ECFCs are a powerful tool to study pathophysiological mechanisms underlying vascular diseases, including von Willebrand disease. In prior research, however, large variations between ECFC lines were observed in, among others, von Willebrand factor (VWF) expression.ObjectiveUnderstand the relation between phenotypic characteristics and VWF‐related parameters of healthy control ECFCs.MethodsECFC lines (n = 16) derived from six donors were studied at maximum cell density. Secreted and intracellular VWF antigen were measured by ELISA. The angiogenic capacity of ECFCs was investigated by the Matrigel tube formation assay. Differences in expression of genes involved in angiogenesis, aging, and endothelial to mesenchymal transition (EndoMT) were measured by quantitative PCR.ResultsDifferent ECFC lines show variable morphologies and cell density at maximum confluency and cell lines with a low maximum cell density show a mixed and more mesenchymal phenotype. We identified a significant positive correlation between maximum cell density and VWF production, both at protein and mRNA level. Also, significant correlations were observed between maximum cell density and several angiogenic, aging and EndoMT parameters.ConclusionsWe observed variations in morphology, maximum cell density, VWF production, and angiogenic potential between healthy control ECFCs. These variations seem to be attributable to differences in aging and EndoMT. Because variations correlate with cell density, we believe that ECFCs maintain a powerful tool to study vascular diseases. It is however important to compare cell lines with the same characteristics and perform experiments at maximum cell density.

Von Willebrand disease (VWD) is the most common inherited bleeding disorder and is mainly caused by dominant-negative mutations in the multimeric protein von Willebrand factor (VWF). These mutations may either result in quantitative or qualitative defects in VWF. VWF is an endothelial protein that is secreted to the circulation upon endothelial activation. Once secreted, VWF multimers bind platelets and chaperone coagulation factor VIII in the circulation. Treatment of VWD focuses on increasing VWF plasma levels, but production and secretion of mutant VWF remain uninterrupted. Presence of circulating mutant VWF might, however, still affect normal hemostasis or functionalities of VWF beyond hemostasis. We hypothesized that inhibition of the production of mutant VWF improves the function of VWF overall and ameliorates VWD phenotypes. We previously proposed the use of allele-specific small-interfering RNAs (siRNAs) that target frequent VWF single nucleotide polymorphisms to inhibit mutant VWF. The aim of this study is to prove the functionality of these allele-specific siRNAs in endothelial colony-forming cells (ECFCs). We isolated ECFCs from a VWD type 2A patient with an intracellular multimerization defect, reduced VWF collagen binding, and a defective processing of proVWF to VWF. After transfection of an allele-specific siRNA that specifically inhibited expression of mutant VWF, we showed amelioration of the laboratory phenotype, with normalization of the VWF collagen binding, improvement in VWF multimers, and enhanced VWF processing. Altogether, we prove that allele-specific inhibition of the production of mutant VWF by siRNAs is a promising therapeutic strategy to improve VWD phenotypes.