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Recycling endosomes maintain plasma membrane homeostasis and are important for cell polarity, migration, and cytokinesis. Yet, the molecular machineries that drive endocytic recycling remain largely unclear. The CORVET complex is a multi-subunit tether required for fusion between early endosomes. Here we show that the CORVET-specific subunits Vps3 and Vps8 also regulate vesicular transport from early to recycling endosomes. Vps3 and Vps8 localise to Rab4-positive recycling vesicles and co-localise with the CHEVI complex on Rab11-positive recycling endosomes. Depletion of Vps3 or Vps8 does not affect transferrin recycling, but delays the delivery of internalised integrins to recycling endosomes and their subsequent return to the plasma membrane. Consequently, Vps3/8 depletion results in defects in integrin-dependent cell adhesion and spreading, focal adhesion formation, and cell migration. These data reveal a role for Vps3 and Vps8 in a specialised recycling pathway important for integrin trafficking. [ABSTRACT FROM AUTHOR]

Integrins are transmembrane αβ glycoproteins that connect the extracellular matrix to the cytoskeleton. The laminin-binding integrin α3β1 is expressed at high levels in lung epithelium and in kidney podocytes. In podocytes, α3β1 associates with the tetraspanin CD151 to maintain a functional filtration barrier. Here, we report on a patient homozygous for a novel missense mutation in the human ITGA3 gene, causing fatal interstitial lung disease and congenital nephrotic syndrome. The mutation caused an alanine-to-serine substitution in the integrin α3 subunit, thereby introducing an N-glycosylation motif at amino acid position 349. We expressed this mutant form of ITGA3 in murine podocytes and found that hyperglycosylation of the α3 precursor prevented its heterodimerization with β1, whereas CD151 association with the α3 subunit occurred normally. Consequently, the β1 precursor accumulated in the ER, and the mutant α3 precursor was degraded by the ubiquitin-proteasome system. Thus, these findings uncover a gain-of-glycosylation mutation in ITGA3 that prevents the biosynthesis of functional α3β1, causing a fatal multiorgan disorder. [ABSTRACT FROM AUTHOR]

Integrin-dependent cell-extracellular matrix adhesion is essential for wound healing, embryonic development, immunity, and tissue organization. Here, we present a protocol for the imaging and quantitative analysis of integrin-dependent cell-matrix adhesions. We describe steps for cell culture; virus preparation; lentiviral transduction; imaging with widefield, confocal, and total internal reflection fluorescence microscopy; and using a script for their quantitative analysis. We then detail procedures for analyzing adhesion dynamics by live-cell imaging and fluorescence recovery after photobleaching (FRAP). For complete details on the use and execution of this protocol, please refer to Margadant et al. (2012), 1 van der Bijl et al. (2020), 2 Amado-Azevedo et al. (2021). 3 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

FNAIT is a pregnancy-associated condition caused by maternal alloantibodies against paternally-inherited platelet antigens, most frequently HPA-1a on integrin β3. The clinical effects range from no symptoms to fatal intracranial hemorrhage, but underlying pathophysiological determinants are poorly understood. Accumulating evidence suggests that differential antibody-Fc-glycosylation, activation of complement/effector cells, and integrin function-blocking effects contribute to clinical outcome. Furthermore, some antibodies preferentially bind platelet integrin αIIbβ3, but others bind αvβ3 on endothelial cells and trophoblasts. Defects in endothelial cells and angiogenesis may therefore contribute to severe anti-HPA-1a associated FNAIT. Moreover, anti-HPA-1a antibodies may cause placental damage, leading to intrauterine growth restriction. We discuss current insights into diversity and actions of HPA-1a antibodies, gathered from clinical studies, in vitro studies, and mouse models. Assessment of all factors determining severity and progression of anti-HPA-1a-associated FNAIT may importantly improve risk stratification and potentially reveal novel treatment strategies, both for FNAIT and other immunohematological disorders., Competing Interests: Declaration of Competing Interest Nothing to declare., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Patients with gray platelet syndrome (GPS) and Neurobeachin-like 2 (NBEAL2) deficiency produce platelets lacking alpha-granules (AGs) and present with lifelong bleeding symptoms. AGs are lysosome-related organelles and store the hemostatic protein von Willebrand factor (VWF) and the transmembrane protein P-selectin. Weibel-Palade bodies (WPBs) are lysosome-related organelles of endothelial cells and also store VWF and P-selectin. In megakaryocytes, NBEAL2 links P-selectin on AGs to the SNARE protein SEC22B on the endoplasmic reticulum, thereby preventing premature release of cargo from AG precursors. In endothelial cells, SEC22B drives VWF trafficking from the endoplasmic reticulum to Golgi and promotes the formation of elongated WPBs, but it is unclear whether this requires NBEAL2., Objectives: To investigate a potential role for NBEAL2 in WPB biogenesis and VWF secretion using NBEAL2-deficient endothelial cells., Methods: The interaction of SEC22B with NBEAL2 in endothelial cells was investigated by interatomic mass spectrometry and pull-down analysis. Endothelial colony forming cells were isolated from healthy controls and 3 unrelated patients with GPS and mutations in NBEAL2 ., Results: We showed that SEC22B binds to NBEAL2 in ECs. Endothelial colony forming cells derived from a patient with GPS are deficient in NBEAL2 but reveal normal formation and maturation of WPBs and normal WPB cargo recruitment. Neither basal nor histamine-induced VWF secretion is altered in the absence of NBEAL2., Conclusions: Although NBEAL2 deficiency causes the absence of AGs in patients with GPS, it does not impact WPB functionality in ECs. Our data highlight the differences in the regulatory mechanisms between these 2 hemostatic storage compartments., (© 2023 The Author(s).)

The endothelium is a dynamic, semipermeable layer lining all blood vessels, regulating blood vessel formation and barrier function. Proper composition and function of the endothelial barrier are required for fluid homeostasis, and clinical conditions characterized by barrier disruption are associated with severe morbidity and high mortality rates. Endothelial barrier properties are regulated by cell-cell junctions and intracellular signaling pathways governing the cytoskeleton, but recent insights indicate an increasingly important role for integrin-mediated cell-matrix adhesion and signaling in endothelial barrier regulation. Here, we discuss diseases characterized by endothelial barrier disruption, and provide an overview of the composition of endothelial cell-matrix adhesion complexes and associated signaling pathways, their crosstalk with cell-cell junctions, and with other receptors. We further present recent insights into the role of cell-matrix adhesions in the developing and mature/adult endothelium of various vascular beds, and discuss how the dynamic regulation and turnover of cell-matrix adhesions regulates endothelial barrier function in (patho)physiological conditions like angiogenesis, inflammation and in response to hemodynamic stress. Finally, as clinical conditions associated with vascular leak still lack direct treatment, we focus on how understanding of endothelial cell-matrix adhesion may provide novel targets for treatment, and discuss current translational challenges and future perspectives.

Collective cell migration is crucial for a variety of pathophysiological processes including embryonic development, wound healing, carcinoma invasion, and sprouting angiogenesis. The behavior of leading and following cells during migration is highly dynamic and involves extensive cellular morphological changes mediated by the actin cytoskeleton. Imaging these rapid and dynamic changes over time requires expression of fluorescent proteins and/or live labeling with fluorescent probes, followed by acquiring series of image stacks at short intervals. This presents significant challenges related to dye cytotoxicity, signal loss, and in particular phototoxicity resulting from repeated irradiation, especially when using separate channels for multiple dyes and when imaging large z-stacks at short time intervals. In this chapter, we present methods for multicolor live-cell labeling of primary human endothelial cell populations, followed by multi-position time-lapse imaging in 2D and in 3D protein matrices. These approaches can be performed in combination with RNA interference to suppress the expression of specific proteins, as well as in mosaic assays using mixtures of differentially labeled cell populations. Finally, we present a protocol for long-term imaging at low laser intensity to minimize laser-induced cell damage, followed by post-imaging signal enhancement using artificial intelligence., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Cell migration plays an essential role in many pathophysiological processes, including embryonic development, wound healing, immunity, and cancer invasion, and is therefore a widely studied phenomenon in many different fields from basic cell biology to regenerative medicine. During the past decades, a multitude of increasingly complex methods have been developed to study cell migration. Here we compile a series of current state-of-the-art methods and protocols to investigate cell migration in a variety of model systems ranging from cells, organoids, tissue explants, and microfluidic systems to Drosophila, zebrafish, and mice. Together they cover processes as diverse as nuclear deformation, energy consumption, endocytic trafficking, and matrix degradation, as well as tumor vascularization and cancer cell invasion, sprouting angiogenesis, and leukocyte extravasation. Furthermore, methods to study developmental processes such as neural tube closure, germ layer specification, and branching morphogenesis are included, as well as scripts for the automated analysis of several aspects of cell migration. Together, this book constitutes a unique collection of methods of prime importance to those interested in the analysis of cell migration., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Von Willebrand factor (VWF) is a glycoprotein that is secreted into the circulation and controls bleeding by promoting adhesion and aggregation of blood platelets at sites of vascular injury. Substantial inter-individual variation in VWF plasma levels exists among the healthy population. Prior to secretion, VWF polymers are assembled and condensed into helical tubules, which are packaged into Weibel-Palade bodies (WPBs), a highly specialized post-Golgi storage compartment in vascular endothelial cells. In the inherited bleeding disorder Von Willebrand disease (VWD), mutations in the VWF gene can cause qualitative or quantitative defects, limiting protein function, secretion, or plasma survival. However, pathogenic VWF mutations cannot be found in all VWD cases. Although an increasing number of genetic modifiers have been identified, even more rare genetic variants that impact VWF plasma levels likely remain to be discovered. Here, we summarize recent evidence that modulation of the early secretory pathway has great impact on the biogenesis and release of WPBs. Based on these findings, we propose that rare, as yet unidentified quantitative trait loci influencing intracellular VWF transport contribute to highly variable VWF levels in the population. These may underlie the thrombotic complications linked to high VWF levels, as well as the bleeding tendency in individuals with low VWF levels., (© 2022 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Von Willebrand factor (VWF) is a multimeric hemostatic protein primarily synthesized in endothelial cells. VWF is stored in endothelial storage organelles, the Weibel-Palade bodies (WPB), whose biogenesis strongly depends on VWF anterograde trafficking and Golgi architecture. Elongated WPB morphology is correlated to longer VWF strings with better adhesive properties. We previously identified the SNARE SEC22B, which is involved in anterograde endoplasmic reticulum-to-Golgi transport, as a novel regulator of WPB elongation. To elucidate novel determinants of WPB morphology we explored endothelial SEC22B interaction partners in a mass spectrometry-based approach, identifying the Golgi SNARE Syntaxin 5 (STX5). We established STX5 knockdown in endothelial cells using shRNA-dependent silencing and analyzed WPB and Golgi morphology, using confocal and electron microscopy. STX5-depleted endothelial cells exhibited extensive Golgi fragmentation and decreased WPB length, which was associated with reduced intracellular VWF levels, and impaired stimulated VWF secretion. However, the secretion-incompetent organelles in shSTX5 cells maintained WPB markers such as Angiopoietin 2, P-selectin, Rab27A, and CD63. In brief, we identified SNARE protein STX5 as a novel regulator of WPB biogenesis.

von Willebrand factor (VWF) is an essential hemostatic protein that is synthesized and secreted by endothelial cells and stored in Weibel-Palade bodies (WPBs). The secretory Rab GTPases Rab27A, Rab3B, and Rab3D have been linked with WPB trafficking and secretion. How these Rabs are activated and recruited to WPBs remains elusive. In this study, we identified MAP kinase-activating death domain (MADD) as the guanine nucleotide exchange factor for Rab27A and both Rab3 isoforms in primary human endothelial cells. Rab activity assays revealed a reduction in Rab27A, Rab3B, and Rab3D activation upon MADD silencing. Rab activation, but not binding, was dependent on the differentially expressed in normal and neoplastic cells (DENN) domain of MADD, indicating the potential existence of 2 Rab interaction modules. Furthermore, immunofluorescent analysis showed that Rab27A, Rab3B, and Rab3D recruitment to WPBs was dramatically decreased upon MADD knockdown, revealing that MADD drives Rab membrane targeting. Artificial mistargeting of MADD using a TOMM70 tag abolished Rab27A localization to WPB membranes in a DENN domain-dependent manner, indicating that normal MADD localization in the cytosol is crucial. Activation of Rab3B and Rab3D was reduced upon Rab27A silencing, suggesting that activation of these Rabs is enhanced through previous activation of Rab27A by MADD. MADD silencing did not affect WPB morphology, but it did reduce VWF intracellular content. Furthermore, MADD-depleted cells exhibited decreased histamine-evoked VWF release, similar to Rab27A-depleted cells. In conclusion, MADD acts as a master regulator of VWF secretion by coordinating the activation and membrane targeting of secretory Rabs to WPBs., (© 2021 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is presenting as a systemic disease associated with vascular inflammation and endothelial injury. Severe forms of SARS-CoV-2 infection induce acute respiratory distress syndrome (ARDS) and there is still an ongoing debate on whether COVID-19 ARDS and its perfusion defect differs from ARDS induced by other causes. Beside pro-inflammatory cytokines (such as interleukin-1 β [IL-1β] or IL-6), several main pathological phenomena have been seen because of endothelial cell (EC) dysfunction: hypercoagulation reflected by fibrin degradation products called D-dimers, micro- and macrothrombosis and pathological angiogenesis. Direct endothelial infection by SARS-CoV-2 is not likely to occur and ACE-2 expression by EC is a matter of debate. Indeed, endothelial damage reported in severely ill patients with COVID-19 could be more likely secondary to infection of neighboring cells and/or a consequence of inflammation. Endotheliopathy could give rise to hypercoagulation by alteration in the levels of different factors such as von Willebrand factor. Other than thrombotic events, pathological angiogenesis is among the recent findings. Overexpression of different proangiogenic factors such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (FGF-2) or placental growth factors (PlGF) have been found in plasma or lung biopsies of COVID-19 patients. Finally, SARS-CoV-2 infection induces an emergency myelopoiesis associated to deregulated immunity and mobilization of endothelial progenitor cells, leading to features of acquired hematological malignancies or cardiovascular disease, which are discussed in this review. Altogether, this review will try to elucidate the pathophysiology of thrombotic complications, pathological angiogenesis and EC dysfunction, allowing better insight in new targets and antithrombotic protocols to better address vascular system dysfunction. Since treating SARS-CoV-2 infection and its potential long-term effects involves targeting the vascular compartment and/or mobilization of immature immune cells, we propose to define COVID-19 and its complications as a systemic vascular acquired hemopathy., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

We describe an optimized, cost-effective, reproducible, and robust protocol to study sprouting angiogenesis in glass-bottom 96-well plates by confocal microscopy, ideal for screening of drug or shRNA libraries. Effective and stable knockdown of gene expression in primary endothelial cells is achieved by lentiviral transduction. Dynamic behavior of individual cells and fluorescent proteins is analyzed by time-lapse imaging, while competitive advantages in tip cell formation are assessed using mixtures of differentially labeled cell populations. Finally, we present a macro for high-throughput analysis. For complete information on the use and execution of this protocol, please refer to van der Bijl et al. (2020) and Kempers et al. (2021)., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

Sprouting angiogenesis is key to many pathophysiological conditions, and is strongly regulated by vascular endothelial growth factor (VEGF) signaling through VEGF receptor 2 (VEGFR2). Here we report that the early endosomal GTPase Rab5C and its activator RIN2 prevent lysosomal routing and degradation of VEGF-bound, internalized VEGFR2 in human endothelial cells. Stabilization of endosomal VEGFR2 levels by RIN2/Rab5C is crucial for VEGF signaling through the ERK and PI3-K pathways, the expression of immediate VEGF target genes, as well as specification of angiogenic 'tip' and 'stalk' cell phenotypes and cell sprouting. Using overexpression of Rab mutants, knockdown and CRISPR/Cas9-mediated gene editing, and live-cell imaging in zebrafish, we further show that endosomal stabilization of VEGFR2 levels is required for developmental angiogenesis in vivo. In contrast, the premature degradation of internalized VEGFR2 disrupts VEGF signaling, gene expression, and tip cell formation and migration. Thus, an endosomal feedforward mechanism maintains receptor signaling by preventing lysosomal degradation, which is directly linked to the induction of target genes and cell fate in collectively migrating cells during morphogenesis., (© 2021. The Author(s).)

Endothelial barrier disruption and vascular leak importantly contribute to organ dysfunction and mortality during inflammatory conditions like sepsis and acute respiratory distress syndrome. We identified the kinase Arg/Abl2 as a mediator of endothelial barrier disruption, but the role of Arg in endothelial monolayer regulation and its relevance in vivo remain poorly understood. Here we show that depletion of Arg in endothelial cells results in the activation of both RhoA and Rac1, increased cell spreading and elongation, redistribution of integrin-dependent cell-matrix adhesions to the cell periphery, and improved adhesion to the extracellular matrix. We further show that Arg is activated in the endothelium during inflammation, both in murine lungs exposed to barrier-disruptive agents, and in pulmonary microvessels of septic patients. Importantly, Arg-depleted endothelial cells were less sensitive to barrier-disruptive agents. Despite the formation of F-actin stress fibers and myosin light chain phosphorylation, Arg depletion diminished adherens junction disruption and intercellular gap formation, by reducing the disassembly of cell-matrix adhesions and cell retraction. In vivo, genetic deletion of Arg diminished vascular leak in the skin and lungs, in the presence of a normal immune response. Together, our data indicate that Arg is a central and non-redundant regulator of endothelial barrier integrity, which contributes to cell retraction and gap formation by increasing the dynamics of adherens junctions and cell-matrix adhesions in a Rho GTPase-dependent fashion. Therapeutic inhibition of Arg may provide a suitable strategy for the treatment of a variety of clinical conditions characterized by vascular leak., (© 2021. The Author(s).)

Integrins are transmembrane receptors that transduce biochemical and mechanical signals across the plasma membrane and promote cell adhesion and migration. In addition, integrin adhesion complexes are functionally and structurally linked to components of the intracellular trafficking machinery and accumulating data now reveal that they are key regulators of endocytosis and exocytosis in a variety of cell types. Here, we highlight recent insights into integrin control of intracellular trafficking in processes such as degranulation, mechanotransduction, cell-cell communication, antibody production, virus entry, Toll-like receptor signaling, autophagy, and phagocytosis, as well as the release and uptake of extracellular vesicles. We discuss the underlying molecular mechanisms and the implications for a range of pathophysiological contexts, including hemostasis, immunity, tissue repair, cancer, and viral infection., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: A range of classification systems are in use for the measurement of nursing workload in Intensive Care Units. However, it is unknown to what extent the measured (objective) nursing workload, usually in terms of the amount of nursing activities, is related to the workload actually experienced (perceived) by nurses., Objectives: The aim of this study was to assess the association between the objective nursing workload and the perceived nursing workload and to identify other factors associated with the perceived nursing workload., Methods: We measured the objective nursing workload with the Nursing Activities Score and the perceived nursing workload with the NASA-Task Load Index during 228 shifts in eight different Intensive Care Units. We used linear mixed-effect regression models to analyze the association between the objective and perceived nursing workload. Furthermore, we investigated the association of patient characteristics (severity of illness, comorbidities, age, body mass index, and planned or unplanned admission), education level of the nurse, and contextual factors (numbers of patients per nurse, the type of shift (day, evening, night) and day of admission or discharge) with perceived nursing workload. We adjusted for confounders., Results: We did not find a significant association between the observed workload per nurse and perceived nursing workload (p=0.06). The APACHE-IV Acute Physiology Score of a patient was significantly associated with the perceived nursing workload, also after adjustment for confounders (p=0.02). None of the other patient characteristics was significantly associated with perceived nursing workload. Being a certified nurse or a student nurse was the only nursing or contextual factor significantly associated with the perceived nursing workload, also after adjustment for confounders (p=0.03)., Conclusion: Workload is perceived differently by nurses compared to the objectively measured workload by the Nursing Activities Score. Both the severity of illness of the patient and being a student nurse are factors that increase the perceived nursing workload. To keep the workload of nurses in balance, planning nursing capacity should be based on the Nursing Activities Score, on the severity of patient illness and the graduation level of the nurse., (Copyright © 2020. Published by Elsevier Ltd.)

Collective cell behaviour during embryogenesis and tissue repair requires the coordination of intercellular junctions, cytoskeleton-dependent shape changes controlled by Rho GTPases, and integrin-dependent cell-matrix adhesion. Many different integrins are simultaneously expressed during wound healing, embryonic development, and sprouting angiogenesis, suggesting that there is extensive integrin/integrin cross-talk to regulate cell behaviour. Here, we show that fibronectin-binding β1 and β3 integrins do not act synergistically, but rather antagonize each other during collective cell processes in neuro-epithelial cells, placental trophoblasts, and endothelial cells. Reciprocal β1/β3 antagonism controls RhoA activity in a kindlin-2-dependent manner, balancing cell spreading, contractility, and intercellular adhesion. In this way, reciprocal β1/β3 antagonism controls cell cohesion and cellular plasticity to switch between extreme and opposing states, including epithelial versus mesenchymal-like phenotypes and collective versus individual cell migration. We propose that integrin/integrin antagonism is a universal mechanism to effectuate social cellular interactions, important for tissue morphogenesis, endothelial barrier function, trophoblast invasion, and sprouting angiogenesis., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2020. Published by Elsevier B.V.)

Major efforts are made to characterize the presence of microRNA (miRNA) and messenger RNA in blood plasma to discover novel disease-associated biomarkers. MiRNAs in plasma are associated to several types of macromolecular structures, including extracellular vesicles (EV), lipoprotein particles (LPP) and ribonucleoprotein particles (RNP). RNAs in these complexes are recovered at variable efficiency by commonly used EV- and RNA isolation methods, which causes biases and inconsistencies in miRNA quantitation. Besides miRNAs, various other non-coding RNA species are contained in EV and present within the pool of plasma extracellular RNA. Members of the Y-RNA family have been detected in EV from various cell types and are among the most abundant non-coding RNA types in plasma. We previously showed that shuttling of full-length Y-RNA into EV released by immune cells is modulated by microbial stimulation. This indicated that Y-RNAs could contribute to the functional properties of EV in immune cell communication and that EV-associated Y-RNAs could have biomarker potential in immune-related diseases. Here, we investigated which macromolecular structures in plasma contain full length Y-RNA and whether the levels of three Y-RNA subtypes in plasma (Y1, Y3 and Y4) change during systemic inflammation. Our data indicate that the majority of full length Y-RNA in plasma is stably associated to EV. Moreover, we discovered that EV from different blood-related cell types contain cell-type-specific Y-RNA subtype ratios. Using a human model for systemic inflammation, we show that the neutrophil-specific Y4/Y3 ratios and PBMC-specific Y3/Y1 ratios were significantly altered after induction of inflammation. The plasma Y-RNA ratios strongly correlated with the number and type of immune cells during systemic inflammation. Cell-type-specific "Y-RNA signatures" in plasma EV can be determined without prior enrichment for EV, and may be further explored as simple and fast test for diagnosis of inflammatory responses or other immune-related diseases., (© 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group on behalf of The International Society for Extracellular Vesicles.)

Vascular endothelial growth factor-A (VEGF-A/VEGF) interaction with VEGF receptor 2 (VEGFR2) is key for sprouting angiogenesis in health and disease. VEGF/VEGFR2 signaling promotes endothelial proliferation and migration, as well as the hierarchical organization into leader (tip) and follower (stalk) cells via a dynamic interplay with Notch. Recent studies reveal novel molecular mechanisms to fine-tune VEGF/Notch signaling and tip/stalk cell function during sprouting angiogenesis.

Fibrillins constitute a family of large extracellular glycoproteins which multimerize to form microfibrils, an important structure in the extracellular matrix. It has long been assumed that fibrillin-2 was barely present during postnatal life, but it is now clear that fibrillin-2 molecules form the structural core of microfibrils, and are masked by an outer layer of fibrillin-1. Mutations in fibrillins give rise to heritable connective tissue disorders, including Marfan syndrome and congenital contractural arachnodactyly. Fibrillins also play an important role in matrix sequestering of members of the transforming growth factor-β family, and in context of Marfan syndrome excessive TGF-β activation has been observed. TGF-β activation is highly dependent on integrin binding, including integrin αvβ8 and αvβ6, which are upregulated upon TGF-β exposure. TGF-β is also involved in tumor progression, metastasis, epithelial-to-mesenchymal transition and tumor angiogenesis. In several highly vascularized types of cancer such as hepatocellular carcinoma, a positive correlation was found between increased TGF-β plasma concentrations and tumor vascularity. Interestingly, fibrillin-1 has a higher affinity to TGF-β and, therefore, has a higher capacity to sequester TGF-β compared to fibrillin-2. The previously reported downregulation of fibrillin-1 in tumor endothelium affects the fibrillin-1/fibrillin-2 ratio in the microfibrils, exposing the normally hidden fibrillin-2. We postulate that fibrillin-2 exposure in the tumor endothelium directly stimulates tumor angiogenesis by influencing TGF-β sequestering by microfibrils, leading to a locally higher active TGF-β concentration in the tumor microenvironment. From a therapeutic perspective, fibrillin-2 might serve as a potential target for future anti-cancer therapies., Competing Interests: Declaration of Competing Interest The authors declare to have no conflict of interest.This work was supported by the KWF Cancer Society [2018–11651]., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Integrins are a large family of heterodimeric cell surface receptors that bind prototypic ligands on neighboring cells or in the extracellular matrix. Numerous studies have revealed key roles for platelet and leukocyte integrins in adhesion and migration and, thereby, their significance for hemostasis and immunity. The clinical importance of these integrins has also become clear, because aberrant integrin expression and/or behavior are associated with bleeding disorders, immunodeficiency, or autoimmune diseases. Importantly, overwhelming evidence gathered over recent years shows that regulation of integrin function is far more complex than previously assumed; a picture has emerged of multiple cytoplasmic, cell surface, and extracellular regulators working together to ensure cell type-specific and integrin-specific control of integrin functions. Here, we discuss recent insights into the dynamic activation and suppression of hematopoietic integrins, as well as their implications for platelet and leukocyte function in health and disease., (© 2020 by The American Society of Hematology.)

Objectives: Studies have shown contradicting results on the association of nursing workload and mortality. Most of these studies expressed workload as patients per nurse ratios; however, this does not take into account that some patients require more nursing time than others. Nursing time can be quantified by tools like the Nursing Activities Score. We investigated the association of the Nursing Activities Score per nurse ratio, respectively, the patients per nurse ratio with in-hospital mortality in ICUs., Design: Retrospective analysis of the National Intensive Care Evaluation database., Setting: Fifteen Dutch ICUs., Patients: All ICU patients admitted to and registered ICU nurses working at 15 Dutch ICUs between January 1, 2016, and January 1, 2018, were included. The association of mean or day 1 patients per nurse ratio and Nursing Activities Score per nurse ratio with in-hospital mortality was analyzed using logistic regression models., Interventions: None., Measurements and Main Results: Nursing Activities Score per nurse ratio greater than 41 for both mean Nursing Activities Score per nurse ratio as well as Nursing Activities Score per nurse ratio on day 1 were associated with a higher in-hospital mortality (odds ratios, 1.19 and 1.17, respectively). After case-mix adjustment the association between a Nursing Activities Score per nurse ratio greater than 61 for both mean Nursing Activities Score per nurse ratio as well as Nursing Activities Score per nurse ratio on day 1 and in-hospital mortality remained significant (odds ratios, 1.29 and 1.26, respectively). Patients per nurse ratio was not associated with in-hospital mortality., Conclusions: A higher Nursing Activities Score per nurse ratio was associated with higher in-hospital mortality. In contrast, no association was found between patients per nurse ratios and in-hospital mortality in The Netherlands. Therefore, we conclude that it is more important to focus on the nursing workload that the patients generate rather than on the number of patients the nurse has to take care of in the ICU.

Background: Synthesis of the hemostatic protein von Willebrand factor (VWF) drives formation of endothelial storage organelles called Weibel-Palade bodies (WPBs). In the absence of VWF, angiogenic and inflammatory mediators that are costored in WPBs are subject to alternative trafficking routes. In patients with von Willebrand disease (VWD), partial or complete absence of VWF/WPBs may lead to additional bleeding complications, such as angiodysplasia. Studies addressing the role of VWF using VWD patient-derived blood outgrowth endothelial cells (BOECs) have reported conflicting results due to the intrinsic heterogeneity of patient-derived BOECs., Objective: To generate a VWF-deficient endothelial cell model using clustered regularly interspaced short palindromic repeats (CRISPR) genome engineering of blood outgrowth endothelial cells., Methods: We used CRISPR/CRISPR-associated protein 9 editing in single-donor cord blood-derived BOECs (cbBOECs) to generate clonal VWF -/- cbBOECs. Clones were selected using high-throughput screening, VWF mutations were validated by sequencing, and cells were phenotypically characterized., Results: Two VWF -/- BOEC clones were obtained and were entirely devoid of WPBs, while their overall cell morphology was unaltered. Several WPB proteins, including CD63, syntaxin-3 and the cargo proteins angiopoietin (Ang)-2, interleukin (IL)-6, and IL-8 showed alternative trafficking and secretion in the absence of VWF. Interestingly, Ang-2 was relocated to the cell periphery and colocalized with Tie-2., Conclusions: CRISPR editing of VWF provides a robust method to create VWF- deficient BOECs that can be directly compared to their wild-type counterparts. Results obtained with our model system confirmed alternative trafficking of several WPB proteins in the absence of VWF and support the theory that increased Ang-2/Tie-2 interaction contributes to angiogenic abnormalities in VWD patients., (© 2019 The Authors. Research and Practice in Thrombosis and Haemostasis published by Wiley Periodicals, Inc on behalf of International Society on Thrombosis and Haemostasis.)

Recycling endosomes maintain plasma membrane homeostasis and are important for cell polarity, migration, and cytokinesis. Yet, the molecular machineries that drive endocytic recycling remain largely unclear. The CORVET complex is a multi-subunit tether required for fusion between early endosomes. Here we show that the CORVET-specific subunits Vps3 and Vps8 also regulate vesicular transport from early to recycling endosomes. Vps3 and Vps8 localise to Rab4-positive recycling vesicles and co-localise with the CHEVI complex on Rab11-positive recycling endosomes. Depletion of Vps3 or Vps8 does not affect transferrin recycling, but delays the delivery of internalised integrins to recycling endosomes and their subsequent return to the plasma membrane. Consequently, Vps3/8 depletion results in defects in integrin-dependent cell adhesion and spreading, focal adhesion formation, and cell migration. These data reveal a role for Vps3 and Vps8 in a specialised recycling pathway important for integrin trafficking.

Background: Reduced muscle density is associated with an increased risk of postoperative complications. We examined the prognostic value of muscle density as a predictor of postoperative complications in elderly patients undergoing surgery for colorectal cancer., Methods: Patients (≥70 years) who underwent surgery for colorectal cancer between 2006 and 2013 were selected from a prospective single centre database. The Hounsfield Unit Average (HUA or HU/mm 2 ) of the psoas muscles at the level of the third lumbar vertebra was calculated on the scan. High and low muscle density groups were identified based on the lowest gender specific HUAC quartile. Major postoperative complications (Clavien-Dindo (CD) ≥3) within 30 days after surgery were retrospectively documented. Logistic regression analysis was used to identify risk factors for postoperative complications., Results: A total of 373 patients (median age = 78 years) were included in this study. The mean muscle density score was 24.5 ± 4.3 HU/mm 2 for males and 26.3 ± 5.0 HU/mm 2 for females. The cut-off point for the lowest gender specific quartile was ≤22.0 HU/mm 2 for males and ≤23.5 HU/mm 2 for females. After multivariable regression, there was a statistically significant association between muscle density and CD ≥ 3 (OR = 1.84 (95% CI 1.11-3.06), p = 0.019). Anastomotic leakage in patients with a primary anastomosis (n = 287) occurred more often in patients with low muscle density (11.7% vs 23.3%, p = 0.016). The associations remained significant after correction for confounders., Conclusion: Low muscle density is associated with major postoperative complications in older patients who undergo surgery for colorectal cancer., (Published by Elsevier Ltd.)

Chloride intracellular channel protein 4 (CLIC4) exists in both soluble and membrane-associated forms, and is implicated in diverse cellular processes, ranging from ion channel formation to intracellular membrane remodeling. CLIC4 is rapidly recruited to the plasma membrane by lysophosphatidic acid (LPA) and serum, suggesting a possible role for CLIC4 in exocytic-endocytic trafficking. However, the function and subcellular target(s) of CLIC4 remain elusive. Here, we show that in HeLa and MDA-MB-231 cells, CLIC4 knockdown decreases cell-matrix adhesion, cell spreading and integrin signaling, whereas it increases cell motility. LPA stimulates the recruitment of CLIC4 to β1 integrin at the plasma membrane and in Rab35-positive endosomes. CLIC4 is required for both the internalization and the serum- or LPA-induced recycling of β1 integrin, but not for EGF receptor trafficking. Furthermore, we show that CLIC4 suppresses Rab35 activity and antagonizes Rab35-dependent regulation of β1 integrin trafficking. Our results define CLIC4 as a regulator of Rab35 activity and serum- and LPA-dependent integrin trafficking., (© 2014. Published by The Company of Biologists Ltd.)

We show that the nanoscale adhesion geometry controls the spreading and differentiation of epidermal stem cells. We find that cells respond to such hard nanopatterns similarly to their behavior on soft hydrogels. Cellular responses were seen to stem from local changes in diffusion dynamics of the adapter protein vinculin and associated impaired mechanotransduction rather than impaired recruitment of proteins involved in focal adhesion formation.

From a forward genetic screen for epidermal defects in zebrafish, we identified a loss-of-function mutation in Kindlin-1, an essential regulator of integrin function. The mutation generates a premature stop codon, deleting the integrin-binding site. The mutant zebrafish develops cell-matrix and cell-cell adhesion defects in the basal epidermis leading to progressive fin rupturing, and was therefore designated rupturing-of-fins (rof). Similar defects were observed in the epidermis of Kindler syndrome patients, carrying a loss-of-function mutation in kindlin-1. Mutational analysis and rescue experiments in zebrafish revealed that residues K610, W612, and I647 in the F3 domain are essential for Kindlin-1 function in vivo, and that Kindlin-2 can functionally compensate for the loss of Kindlin-1. The fin phenotype of rof/kindlin-1 mutants resembles that of badfin mutants, carrying a mutation in integrin α3. We show here that this mutation impairs the biosynthesis of integrin α3β1 and causes cell-matrix and cell-cell defects in vivo. Whereas both Integrin-linked kinase (Ilk) and Kindlin-1 cooperate with Integrin α3β1 to resist trauma-induced epidermal defects, Kindlin-1 and Ilk, surprisingly, do not act synergistically but in parallel. Thus, the rof/kindlin-1 mutant zebrafish provides a unique model system to study epidermal adhesion mechanisms in vivo.

Loss-of-function mutations in the gene encoding the integrin co-activator kindlin-1 cause Kindler syndrome. We report a novel kindlin-1-deficient keratinocyte cell line derived from a Kindler syndrome patient. Despite the expression of kindlin-2, the patient's cells display several hallmarks related to reduced function of β1 integrins, including abnormal cell morphology, cell adhesion, cell spreading, focal adhesion assembly, and cell migration. Defective cell adhesion was aggravated by kindlin-2 depletion, indicating that kindlin-2 can compensate to a certain extent for the loss of kindlin-1. Intriguingly, β1 at the cell-surface was aberrantly glycosylated in the patient's cells, and its expression was considerably reduced, both in cells in vitro and in the patient's epidermis. Reconstitution with wild-type kindlin-1 but not with a β1-binding defective mutant restored the aberrant β1 expression and glycosylation, and normalized cell morphology, adhesion, spreading, and migration. Furthermore, the expression of wild-type kindlin-1, but not of the integrin-binding-defective mutant, increased the stability of integrin-mediated cell-matrix adhesions and enhanced the redistribution of internalized integrins to the cell surface. Thus, these data uncover a role for kindlin-1 in the regulation of integrin trafficking and adhesion turnover.

Integrin-mediated cytoskeletal tension supports growth-factor-induced proliferation, and disruption of the actin cytoskeleton in growth factor-stimulated cells prevents the re-expression of cyclin D and cell cycle re-entry from quiescence. In contrast to cells that enter the cell cycle from G0, cycling cells continuously express cyclin D, and are subject to major cell shape changes during the cell cycle. Here, we investigated the cell cycle requirements for cytoskeletal tension and cell spreading in cycling mammalian cells that enter G1-phase from mitosis. Disruption of the actin cytoskeleton at progressive time-points in G1-phase induced cell rounding, FA disassembly, and attenuated both integrin signaling and growth factor-induced p44/p42 mitogen-activated protein kinase activation. Although cyclin D expression was reduced, the expression of cyclin A and entry into S-phase were not affected. Moreover, expression of cyclin B1, progression through G2- and M-phase, and commitment to a new cell cycle occurred normally. In contrast, cell cycle progression was strongly prevented by inhibition of MAPK activity in G1-phase, whereas cell spreading, cytoskeletal organization, and integrin signaling were not impaired. MAPK inhibition also prevented cytoskeleton-independent cell cycle progression. Thus, these results uncouple the requirements for cell spreading and cytoskeletal organization from MAPK signaling, and show that cycling mammalian cells can proliferate independently of actin stress fibers, focal adhesions, or cell spreading, as long as a threshold level of MAPK activity is sustained.

Introduction of the integrin β1- but not the β3-subunit in GE11 cells induces an epithelial-to-mesenchymal-transition (EMT)-like phenomenon that is characterized by the loss of cell-cell contacts, cell scattering, increased cell migration and RhoA activity, and fibronectin fibrillogenesis. Because galactose-binding lectins (galectins) have been implicated in these phenomena, we investigated whether galectins are involved in the β1-induced phenotype. We examined 9 galectins and, intriguingly, found that the expression of galectin-3 (Gal-3) is specifically induced by β1 but not by β3. Using β1-β3 chimeric integrins, we show that the induction of Gal-3 expression requires the hypervariable region in the extracellular domain of β1, but not its cytoplasmic tail. Furthermore, Gal-3 expression does not depend on RhoA signaling, serum factors, or any of the major signal transduction pathways involving protein kinase C (PKC), p38 mitogen-activated protein kinase (p38MAPK), extracellular signal-regulated kinase-1/-2 (ERK-1/2), phosphatidylinositol-3-OH kinase (PI3-K), or Src kinases. Instead, Gal-3 expression is controlled in an epigenetic manner. Whereas DNA methylation of the Lgals3 promoter maintains Gal-3 silencing in GE11 cells, expression of β1 causes its demethylation, leading to transcriptional activation of the Lgals3 gene. In turn, Gal-3 expression enhances β1 integrin-mediated cell adhesion to fibronectin (FN) and laminin (LN), as well as cell migration. Gal-3 also promotes β1-mediated cell adhesion to LN and Collagen-1 (Col)-1 in cells that endogenously express Gal-3 and β1 integrins. In conclusion, we identify a functional feedback-loop between β1 integrins and Gal-3 that involves the epigenetic induction of Gal-3 expression during integrin-induced EMT and cell scattering.

Background: Integrins are heterodimeric αβ transmembrane receptors that play key roles in cellular physiology and pathology. Accumulating data indicate that the two NPxY motifs in the cytoplasmic domain of the β1 subunit synergistically promote integrin activation through the binding of talin and kindlin. However, it is unclear how the individual motifs regulate integrin function and trafficking., Results: To investigate how the two NPxY motifs individually control integrin α5β1 function and trafficking, we introduced Y > A mutations in either motif. Disruption of the membrane-proximal NPxY completely prevented α5β1-induced morphological changes, cell scattering and migration, and fibronectin fibrillogenesis. In addition, it reduced α5β1 internalization but not its recycling. In contrast, disruption of the membrane-distal NPxY promoted degradation of α5β1 in late endosomes/lysosomes but did not prevent α5β1-dependent cell scattering, migration, or fibronectin fibrillogenesis. Whereas depletion of either talin-1 or kindlin-2 reduced α5β1 binding to fibronectin and cell adhesion, talin-1 depletion recapitulated the loss-of-function phenotype of the membrane-proximal NPxY mutation, whereas kindlin-2 depletion induced α5β1 accumulation in lysosomes and degradation., Conclusions: The two NPxY motifs of β1 play distinct and separable roles in controlling the function and trafficking of α5β1. Whereas talin binding to the membrane-proximal NPxY is crucial for connecting α5β1 to the actin cytoskeleton and thus permit the tension required for fibronectin fibrillogenesis and cell migration, kindlin binding to the membrane-distal NPxY is dispensable for these events but regulates α5β1 surface expression and degradation., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Integrin adhesion receptors are essential for the normal function of most multicellular organisms, and defective integrin activation or integrin signaling is associated with an array of pathological conditions. Integrins are regulated by conformational changes, clustering, and trafficking, and regulatory mechanisms differ strongly between individual integrins and between cell types. Whereas integrins in circulating blood cells are activated by an inside-out-induced conformational change that favors high-affinity ligand binding, β1-integrins in adherent cells can be activated by force or clustering. In addition, endocytosis and recycling play an important role in the regulation of integrin turnover and integrin redistribution in adherent cells, especially during dynamic processes such as cell migration and invasion. Integrin trafficking is strongly regulated by their cytoplasmic tails, and the mechanisms are now being identified., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The skin forms a barrier against the environment and protects us from mechanical trauma, pathogens, radiation, dehydration, and dangerous temperature fluctuations. The epithelium of the skin, the epidermis, is in a continuous equilibrium of growth and differentiation and has the remarkable capacity to self-renew completely, which relies on reservoirs of stem cells. Epidermal homeostasis is further dependent on proper repair after injury, and on tight adhesion to the underlying basement membrane. Epidermal adhesion is mediated primarily by integrins, cell-surface receptors that connect the extracellular matrix to the cytoskeleton. In addition, numerous in vitro reports have implicated integrins, integrin-associated proteins, or downstream integrin effectors in the regulation of a plethora of cellular processes other than adhesion. Over the past decade, a wealth of information on the function of these proteins has been gathered both from (conditional) knockout mice and from human skin disorders, allowing for a reconstruction of integrin signaling pathways in vivo. Here, we address how epidermal integrins and integrin-associated proteins regulate keratinocyte adhesion, proliferation, and differentiation, as well as signal transduction, re-epithelialization during wound healing, hair growth, and stem cell maintenance. Furthermore, we discuss human pathologies associated with altered integrin functions in the epidermis.

Accumulating evidence indicates that there is extensive crosstalk between integrins and TGF-beta signalling. TGF-beta affects integrin-mediated cell adhesion and migration by regulating the expression of integrins, their ligands and integrin-associated proteins. Conversely, several integrins directly control TGF-beta activation. In addition, a number of integrins can interfere with both Smad-dependent and Smad-independent TGF-beta signalling in different ways, including the regulation of the expression of TGF-beta signalling pathway components, the physical association of integrins with TGF-beta receptors and the modulation of downstream effectors. Reciprocal TGF-beta-integrin signalling is implicated in normal physiology, as well as in a variety of pathological processes including systemic sclerosis, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease and cancer; thus, integrins could provide attractive therapeutic targets to interfere with TGF-beta signalling in these processes.

Re-epithelialization after skin wounding requires both migration and hyperproliferation of keratinocytes. Laminin-332 is deposited during migration over the provisional matrix. To investigate the function of the laminin-332 binding integrin alpha3beta1 in wound re-epithelialization, we generated Itga3flox/flox; K14-Cre mice lacking the alpha3 subunit specifically in the basal layer of the epidermis. These mice are viable but display several skin defects, including local inflammation, hair loss, basement membrane duplication and microblistering at the dermal-epidermal junction, whereas hemidesmosome assembly and keratinocyte differentiation are not impaired. Wound healing is slightly faster in the absence of integrin alpha3beta1, whereas proliferation, the distribution of other integrins and the deposition of basement membrane proteins in the wound bed are unaltered. In vitro, cell spreading is rescued by increased surface expression of alpha6beta1 integrin in the absence of integrin alpha3. The alpha3-deficient keratinocytes migrate with an increased velocity and persistence, whereas proliferation, growth factor signaling, hemidesmosome assembly, and laminin-332 deposition appeared to be normal. We suggest that integrin alpha3beta1 delays keratinocyte migration during wound re-epithelialization, by binding to the laminin-332 that is newly deposited on the wound bed.

Hemidesmosomes (HDs) promote the stable adhesion of basal epithelial cells to the underlying basement membrane (BM). Critical for the mechanical stability of the HD is the interaction between integrin alpha6beta4 and plectin, which is destabilized when HD disassembly is required, for instance, to allow keratinocyte migration during wound healing. Growth factors such as epidermal growth factor (EGF) can trigger HD disassembly and induce phosphorylation of the beta4 intracellular domain. Whereas tyrosine phosphorylation appears to mediate cooperation with growth factor signaling pathways and invasion in carcinoma cells, serine phosphorylation seems the predominant mechanism for regulating HD destabilization. Here, we discuss recent advances that shed light on the residues involved, the identity of the kinases that phosphorylate them, and the interactions that become disrupted by these phosphorylations.

Hemidesmosomes (HDs) are multiprotein adhesion complexes that promote attachment of epithelial cells to the basement membrane. The binding of alpha6beta4 to plectin plays a central role in their assembly. We have defined three regions on beta4 that together harbor all the serine and threonine phosphorylation sites and show that three serines (S1356, S1360, and S1364), previously implicated in HD regulation, prevent the interaction of beta4 with the plectin actin-binding domain when phosphorylated. We have also established that epidermal growth factor receptor activation, which is known to function upstream of HD disassembly, results in the phosphorylation of only one or more of these three residues and the partial disassembly of HDs in keratinocytes. Additionally, we show that S1360 and S1364 of beta4 are the only residues phosphorylated by PKC and PKA in cells, respectively. Taken together, our studies indicate that multiple kinases act in concert to breakdown the structural integrity of HDs in keratinocytes, which is primarily achieved through the phosphorylation of S1356, S1360, and S1364 on the beta4 subunit.