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Pulmonary endothelial cells (ECs) are an essential component of the gas exchange machinery of the lung alveolus. Despite this, the extent and function of lung EC heterogeneity remains incompletely understood. Using single-cell analytics, we identify multiple EC populations in the mouse lung, including macrovascular endothelium (maEC), microvascular endothelium (miECs), and a new population we have termed Car4-high ECs. Car4-high ECs express a unique gene signature, and ligand-receptor analysis indicates they are primed to receive reparative signals from alveolar type I cells. After acute lung injury, they are preferentially localized in regenerating regions of the alveolus. Influenza infection reveals the emergence of a population of highly proliferative ECs that likely arise from multiple miEC populations and contribute to alveolar revascularization after injury. These studies map EC heterogeneity in the adult lung and characterize the response of novel EC subpopulations required for tissue regeneration after acute lung injury. Animal lungs are filled with tiny air sacks called alveoli, where the gas exchanges that keep organisms alive can take place. Small blood vessels known as capillaries come in close contact with the alveoli, allowing oxygen to be extracted from the air into the blood, and carbon dioxide to be released from the blood into the air. The cells that line the inside of these capillaries (known as pulmonary endothelial cells) are important actors in these exchanges. After having been damaged, for example by viruses like influenza, the lungs need to regenerate and create new capillaries. Yet, it was still unclear how pulmonary endothelial cells participate in the healing process, and if capillaries contain several populations of endothelial cells that play different roles. To investigate this question, Niethamer et al. used an approach called single-cell analytics to examine individual endothelial cells in the alveoli of mice infected with influenza. This revealed that different subtypes of endothelial cells exist in capillaries, and that some may be able to perform slightly different jobs during lung recovery. Niethamer et al. found that all subtypes could quickly multiply after injury to create more endothelial cells and re-establish gas exchanges. However, one newly identified group (called Car4-high ECs) was particularly primed to receive orders from damaged alveoli. These cells were also often found at the sites where the alveoli were most injured. Lung injuries are a major cause of death worldwide. Understanding how pulmonary endothelial cells work when the organ is both healthy and injured should help to find ways to boost repair, and to create therapies that could target these cells.

During mammalian development, a subpopulation of endothelial cells in the cardinal vein (CV) expresses lymphatic‐specific genes and subsequently develops into the first lymphatic structures, collectively termed as lymph sacs. Budding, sprouting and ballooning of lymphatic endothelial cells (LECs) have been proposed to underlie the emergence of LECs from the CV, but the exact mechanisms of lymph vessel formation remain poorly understood. Applying selective plane illumination‐based ultramicroscopy to entire wholemount‐immunostained mouse embryos, we visualized the complete developing vascular system with cellular resolution. Here, we report emergence of the earliest detectable LECs as strings of loosely connected cells between the CV and superficial venous plexus. Subsequent aggregation of LECs resulted in formation of two distinct, previously unidentified lymphatic structures, the dorsal peripheral longitudinal lymphatic vessel (PLLV) and the ventral primordial thoracic duct (pTD), which at later stages formed a direct contact with the CV. Providing new insights into their function, we found vascular endothelial growth factor C (VEGF‐C) and the matrix component CCBE1 indispensable for LEC budding and migration. Altogether, we present a significantly more detailed view and novel model of early lymphatic development. Ultramicroscopy of wholemount mouse embryos uncovers the first, previously unknown lymphatic structures in mammals: the dorsal longitudinal lymphatic vessel and the ventral primordial thoracic duct, which eventually connect with the cardinal vein as previously described.

Angiopoietins 1–4 (Ang1–4) represent an important family of growth factors, whose activities are mediated through the tyrosine kinase receptors, Tie1 and Tie2. The best characterized are angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2). Ang1 is a potent angiogenic growth factor signaling through Tie2, whereas Ang2 was initially identified as a vascular disruptive agent with antagonistic activity through the same receptor. Recent data demonstrates that Ang2 has context-dependent agonist activities. Ang2 plays important roles in physiological processes and the deregulation of its expression is characteristic of several diseases. In this review, we summarize the activity of Ang2 on blood and lymphatic endothelial cells, its significance in human physiology and disease, and provide a current view of the molecular signaling pathways regulated by Ang2 in endothelial cells.

The glycocalyx is an extracellular layer lining the lumen of the vascular endothelium, protecting the endothelium from shear stress and atherosclerosis and contributes to coagulation, immune response and microvascular perfusion. The GlycoCheck system estimates glycocalyx' thickness in vessels under the tongue from perfused boundary region (PBR) and microvascular perfusion (red blood cell (RBC) filling) via a camera and dedicated software. Evaluating reproducibility and influence of examination conditions on measurements with the GlycoCheck system. Open, randomised, controlled study including 42 healthy smokers investigating day-to-day, side-of-tongue, inter-investigator variance, intraclass-correlation (ICC) and influence of examination conditions at intervals from 0-180 minutes on PBR and RBC filling. Mean (SD) age was 24.9 (6.1) years, 52% were male. There was no significant intra- or inter-investigator variation for PBR or RBC filling nor for PBR for side-of-tongue. A small day-to-day variance was found for PBR (0.012μm, p = 0.007) and RBC filling (0.003%, p = 0.005) and side-of-tongue, RBC filling (0.025%, p = 0.009). ICC was modest but highly improved by increasing measurements. Small significant influence of cigarette smoking (from 40-180 minutes), high calorie meal intake and coffee consumption was found. The latter two peaking immediately and tapering off but remained significant up to 180 minutes, highest PBR changes for the three being 0.042μm (p<0.05), 0.183μm (p<0.001) and 0.160μm (p<0.05) respectively. Measurements with the GlycoCheck system have a moderate reproducibility, but highly increases with multiple measurements and a small day-to-day variability. Smoking, meal and coffee intake had effects up to 180 minutes, abstinence is recommended at least 180 minutes before GlycoCheck measurements. Future studies should standardise conditions during measurements.

Caveolae are plasma membrane invaginations that may play an important role in numerous cellular processes including transport, signaling, and tumor suppression. By targeted disruption of caveolin-1, the main protein component of caveolae, we generated mice that lacked caveolae. The absence of this organelle impaired nitric oxide and calcium signaling in the cardiovascular system, causing aberrations in endothelium-dependent relaxation, contractility, and maintenance of myogenic tone. In addition, the lungs of knockout animals displayed thickening of alveolar septa caused by uncontrolled endothelial cell proliferation and fibrosis, resulting in severe physical limitations in caveolin-1-disrupted mice. Thus, caveolin-1 and caveolae play a fundamental role in organizing multiple signaling pathways in the cell.

Across the animal kingdom, gastrulation represents a key developmental event during which embryonic pluripotent cells diversify into lineage-specific precursors that will generate the adult organism. Here we report the transcriptional profiles of 116,312 single cells from mouse embryos collected at nine sequential time points ranging from 6.5 to 8.5 days post-fertilization. We construct a molecular map of cellular differentiation from pluripotency towards all major embryonic lineages, and explore the complex events involved in the convergence of visceral and primitive streak-derived endoderm. Furthermore, we use single-cell profiling to show that Tal1 −/− chimeric embryos display defects in early mesoderm diversification, and we thus demonstrate how combining temporal and transcriptional information can illuminate gene function. Together, this comprehensive delineation of mammalian cell differentiation trajectories in vivo represents a baseline for understanding the effects of gene mutations during development, as well as a roadmap for the optimization of in vitro differentiation protocols for regenerative medicine. Single-cell profiling is used to create a molecular-level atlas of cell differentiation trajectories during gastrulation and early organogenesis in the mouse.

To evaluate whether bisphosphonates modulate vascular calcification by a modification in endothelial progenitor cells (EPCs) coexpressing osteoblastic surface markers and genes. We performed a double-blind, randomized study of 20 healthy, early postmenopausal women (from February 1, 2008, through July 31, 2008) treated with placebo or risedronate sodium (35 mg/wk) for 4 months. Peripheral blood was collected at baseline and 4 months to determine serum inflammatory markers, osteoprotegerin, and receptor activator of nuclear factor–κB ligand levels and bone turnover markers. Peripheral blood mononuclear cells were stained for EPC surface markers (CD34, CD133, and vascular endothelial growth factor receptor/kinase insert domain receptor) and osteoblast markers (osteocalcin, alkaline phosphatase, and Stro-1). Risedronate treatment resulted in a significant down-regulation of gene sets for osteoblast differentiation and proliferation in EPCs with a trend of decreasing EPCs coexpressing osteocalcin. Our findings indicate that bisphosphonate treatment down-regulates the expression of osteogenic genes in EPCs and suggest a possible mechanism by which bisphosphonates may inhibit vascular calcification.

Exosomes and other small extracellular vesicles (sEVs) provide a unique mode of cell-to-cell communication in which microRNAs (miRNAs) produced and released from one cell are taken up by cells at a distance where they can enact changes in gene expression 1 – 3 . However, the mechanism by which miRNAs are sorted into exosomes/sEVs or retained in cells remains largely unknown. Here we demonstrate that miRNAs possess sorting sequences that determine their secretion in sEVs (EXOmotifs) or cellular retention (CELLmotifs) and that different cell types, including white and brown adipocytes, endothelium, liver and muscle, make preferential use of specific sorting sequences, thus defining the sEV miRNA profile of that cell type. Insertion or deletion of these CELLmotifs or EXOmotifs in a miRNA increases or decreases retention in the cell of production or secretion into exosomes/sEVs. Two RNA-binding proteins, Alyref and Fus, are involved in the export of miRNAs carrying one of the strongest EXOmotifs, CGGGAG. Increased miRNA delivery mediated by EXOmotifs leads to enhanced inhibition of target genes in distant cells. Thus, this miRNA code not only provides important insights that link circulating exosomal miRNAs to tissues of origin, but also provides an approach for improved targeting in RNA-mediated therapies. MicroRNAs encode sorting sequences that determine whether they are secreted in exosomal vesicles to regulate gene expression in distant cells or retained in cells that produced them, with different sequences used by individual cell types.

The mammalian immune system implements a remarkably effective set of mechanisms for fighting pathogens 1 . Its main components are haematopoietic immune cells, including myeloid cells that control innate immunity, and lymphoid cells that constitute adaptive immunity 2 . However, immune functions are not unique to haematopoietic cells, and many other cell types display basic mechanisms of pathogen defence 3 – 5 . To advance our understanding of immunology outside the haematopoietic system, here we systematically investigate the regulation of immune genes in the three major types of structural cells: epithelium, endothelium and fibroblasts. We characterize these cell types across twelve organs in mice, using cellular phenotyping, transcriptome sequencing, chromatin accessibility profiling and epigenome mapping. This comprehensive dataset revealed complex immune gene activity and regulation in structural cells. The observed patterns were highly organ-specific and seem to modulate the extensive interactions between structural cells and haematopoietic immune cells. Moreover, we identified an epigenetically encoded immune potential in structural cells under tissue homeostasis, which was triggered in response to systemic viral infection. This study highlights the prevalence and organ-specific complexity of immune gene activity in non-haematopoietic structural cells, and it provides a high-resolution, multi-omics atlas of the epigenetic and transcriptional networks that regulate structural cells in the mouse. Structural cells implement a broad range of immune-regulatory functions beyond their roles as barriers and connective tissues, and they utilize an epigenetically encoded potential for immune gene activation in their rapid response to viral infection.

Vascular endothelial growth factor receptor‐3 (VEGFR‐3/Flt4) binds two known members of the VEGF ligand family, VEGF‐C and VEGF‐D, and has a critical function in the remodelling of the primary capillary vasculature of midgestation embryos. Later during development, VEGFR‐3 regulates the growth and maintenance of the lymphatic vessels. In the present study, we have isolated and cultured stable lineages of blood vascular and lymphatic endothelial cells from human primary microvascular endothelium by using antibodies against the extracellular domain of VEGFR‐3. We show that VEGFR‐3 stimulation alone protects the lymphatic endothelial cells from serum deprivation‐induced apoptosis and induces their growth and migration. At least some of these signals are transduced via a protein kinase C‐dependent activation of the p42/p44 MAPK signalling cascade and via a wortmannin‐sensitive induction of Akt phosphorylation. These results define the critical role of VEGF‐C/VEGFR‐3 signalling in the growth and survival of lymphatic endothelial cells. The culture of isolated lymphatic endothelial cells should now allow further studies of the molecular properties of these cells.