Background: Astrocytes play a crucial, yet not fully elucidated role in the selective motor neuron pathology in amyotrophic lateral sclerosis (ALS). Among other responsibilities, astrocytes provide important neuronal homeostatic support, however this function is highly compromised in ALS. The establishment of fully human coculture systems can be used to further study the underlying mechanisms of the dysfunctional intercellular interplay, and has the potential to provide a platform for revealing novel therapeutic entry points.Methods: In this study, we characterised human induced pluripotent stem cell (hiPSC)-derived astrocytes fromFUS-ALS patients, and incorporated these cells into a human motor unit microfluidics model to investigate the astrocytic effect on hiPSC-derived motor neuron network and functional neuromuscular junctions (NMJs) using immunocytochemistry and live-cell recordings.FUS-ALS cocultures were systematically compared to their CRISPR-Cas9 gene-edited isogenic control systems.Results: We observed a dysregulation of astrocyte homeostasis, which resulted in aFUS-ALS-mediated increase in reactivity and secretion of inflammatory cytokines. Upon coculture with motor neurons and myotubes, we detected a cytotoxic effect on motor neuron-neurite outgrowth, NMJ formation and functionality, which was improved or fully rescued by isogenic control astrocytes. We demonstrate that ALS astrocytes have both a gain-of-toxicity and loss-of-support function involving the WNT/β-catenin pathway, ultimately contributing to the disruption of motor neuron homeostasis, intercellular networks and NMJs.Conclusions: Our findings shine light on a complex, yet highly important role of astrocytes in ALS, and provides further insight in to their pathological mechanisms.Graphical

Mutations in the N-terminal WD40 domain of coatomer protein complex subunit α (COPA) cause a type I interferonopathy, typically characterized by alveolar hemorrhage, arthritis, and nephritis. We described 3 heterozygous mutations in the C-terminal domain (CTD) of COPA (p.C1013S, p.R1058C, and p.R1142X) in 6 children from 3 unrelated families with a similar syndrome of autoinflammation and autoimmunity. We showed that these CTD COPA mutations disrupt the integrity and the function of coat protein complex I (COPI). In COPA^R1142X and COPA^R1058C fibroblasts, we demonstrated that COPI dysfunction causes both an anterograde ER-to-Golgi and a retrograde Golgi-to-ER trafficking defect. The disturbed intracellular trafficking resulted in a cGAS/STING-dependent upregulation of the type I IFN signaling in patients and patient-derived cell lines, albeit through a distinct molecular mechanism in comparison with mutations in the WD40 domain of COPA. We showed that CTD COPA mutations induce an activation of ER stress and NF-κB signaling in patient-derived primary cell lines. These results demonstrate the importance of the integrity of the CTD of COPA for COPI function and homeostatic intracellular trafficking, essential to ER homeostasis. CTD COPA mutations result in disease by increased ER stress, disturbed intracellular transport, and increased proinflammatory signaling.

Background: Chronic pressure overload predisposes to heart failure, but the pathogenic role of microvascular endothelial cells (MiVEC) remains unknown. We characterized transcriptional, metabolic, and functional adaptation of cardiac MiVEC to pressure overload in mice and patients with aortic stenosis (AS). Methods: In Tie2-Gfp mice subjected to transverse aortic constriction or sham surgery, we performed RNA sequencing of isolated cardiac Gfp ⁺ -MiVEC and validated the signature in freshly isolated MiVEC from left ventricle outflow tract and right atrium of patients with AS. We next compared their angiogenic and metabolic profiles and finally correlated molecular and pathological signatures with clinical phenotypes of 42 patients with AS (50% women). Results: In mice, transverse aortic constriction induced progressive systolic dysfunction, fibrosis, and reduced microvascular density. After 10 weeks, 25 genes predominantly involved in matrix-regulation were >2-fold upregulated in isolated MiVEC. Increased transcript levels of Cartilage Intermediate Layer Protein ( Cilp ), Thrombospondin-4 , Adamtsl-2 , and Collagen1a1 were confirmed by quantitative reverse transcription polymerase chain reaction and recapitulated in left ventricle outflow tract-derived MiVEC of AS ( P 2-fold in left ventricle outflow tract-MiVEC, proline content by 130% (median, IQR, 58%–474%; P =0.008) and procollagen secretion by 85% (mean [95% CI, 16%–154%]; P <0.05 versus right atrium-MiVEC for all). The altered transcriptome in left ventricle outflow tract-MiVEC was associated with impaired 2-dimensional-vascular network formation and 3-dimensional-spheroid sprouting ( P <0.05 versus right atrium-MiVEC), profibrotic ultrastructural changes, and impaired diastolic left ventricle function, capillary density and functional status, especially in female AS. Conclusions: Pressure overload induces major transcriptional and metabolic adaptations in cardiac MiVEC resulting in excess interstitial fibrosis and impaired angiogenesis. Molecular rewiring of MiVEC is worse in women, compromises functional status, and identifies novel targets for intervention.

Background: Infective endocarditis (IE) is characterized by an infected thrombus at the heart valves. How bacteria bypass the immune system and cause these thrombi remains unclear. Neutrophils releasing NETs (neutrophil extracellular traps) lie at this interface between host defense and coagulation. We aimed to determine the role of NETs in IE immunothrombosis. Methods: We used a murine model of Staphylococcus aureus endocarditis in which IE is provoked on inflamed heart valves and characterized IE thrombus content by immunostaining identifying NETs. Antibody-mediated neutrophil depletion and neutrophil-selective PAD4 (peptidylarginine deiminase 4)-knockout mice were used to clarify the role of neutrophils and NETs, respectively. S. aureus mutants deficient in key virulence factors related to immunothrombosis (nucleases or staphylocoagulases) were investigated. Results: Neutrophils releasing NETs were present in infected thrombi and within cellular infiltrates in the surrounding vasculature. Neutrophil depletion increased occurrence of IE, whereas neutrophil-selective impairment of NET formation did not alter IE occurrence. Absence of S. aureus nuclease, which degrades NETs, did not affect endocarditis outcome. In contrast, absence of staphylocoagulases (coagulase and von Willebrand factor binding protein) led to improved survival, decreased bacteremia, smaller infiltrates, and decreased tissue destruction. Significantly more NETs were present in these vegetations, which correlated with decreased bacteria and cell death in the adjacent vascular wall. Conclusions: Neutrophils protect against IE independent of NET release. Absence of S. aureus coagulases, but not nucleases, reduced IE severity and increased NET levels. Staphylocoagulase-induced fibrin likely hampers NETs from constraining infection and the resultant tissue damage, a hallmark of valve destruction in IE.

Polycaprolactone (PCL) is a promising material for the fabrication of alternatives to autologous grafts used in coronary bypass surgery. PCL biodegrades over time, allowing cells to infiltrate the polymeric matrix, replacing the biodegrading graft, and creating a fully functional vessel constituted of autologous tissue. However, the high hydrophobicity of PCL is associated with poor cell affinity. Surface modification of PCL can increase this cell affinity, making PCL an improved scaffold material for acellular vascular grafts. In this study, the surface of PCL films was modified by hydrolysis, aminolysis, and the combination thereof to introduce carboxyl, hydroxyl, and amino groups on the surface. Only the hydrolyzed films exhibited a significant increase in their hydrophilicity, although further testing showed that all aminolysis conditions had amino groups on the surface. Furthermore, in vitro experiments with human umbilical endothelial cells (HUVECs) were performed to assess changes in cell affinity for PCL due to the surface treatments. PCL treated with sodium hydroxide (NaOH), a hydrolysis reaction, showed a significant increase in endothelial cell adhesion after 24hours with a significant increase in cell survival after 72hours. Thus, NaOH treatment improves the biocompatibility and endothelialization of PCL, creating a competent candidate for artificial, acellular, biodegradable vascular grafts.