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Salvianolic acid B (Sal B) is one of the most bioactive components of Salvia miltiorrhiza, a traditional Chinese herbal medicine that has been commonly used for prevention and treatment of cerebrovascular disorders. However, the mechanism responsible for such protective effects remains largely unknown. It has been considered that cerebral endothelium apoptosis caused by reactive oxygen species including hydrogen peroxide (H(2)O(2)) is implicated in the pathogenesis of cerebrovascular disorders. By examining the effect of Sal B on H(2)O(2)-induced apoptosis in rat cerebral microvascular endothelial cells (rCMECs), we found that Sal B pretreatment significantly attenuated H(2)O(2)-induced apoptosis in rCMECs. We next examined the signaling cascade(s) involved in Sal B-mediated anti-apoptotic effects. We showed that H(2)O(2) induces rCMECs apoptosis mainly through the PI3K/ERK pathway, since a PI3K inhibitor (LY294002) blocked ERK activation caused by H(2)O(2 )and a specific inhibitor of MEK (U0126) protected cells from apoptosis. On the other hand, blockage of the PI3K/Akt pathway abrogated the protective effect conferred by Sal B and potentated H(2)O(2)-induced apoptosis, suggesting that Sal B prevents H(2)O(2)-induced apoptosis predominantly through the PI3K/Akt (upstream of ERK) pathway. Our findings provide the first evidence that H(2)O(2) induces rCMECs apoptosis via the PI3K/MEK/ERK pathway and that Sal B protects rCMECs against H(2)O(2)-induced apoptosis through the PI3K/Akt/Raf/MEK/ERK pathway.

The vascular endothelium, a monolayer of endothelial cells (EC), constitutes the inner cellular lining of arteries, veins and capillaries and therefore is in direct contact with the components and cells of blood. The endothelium is not only a mere barrier between blood and tissues but also an endocrine organ. It actively controls the degree of vascular relaxation and constriction, and the extravasation of solutes, fluid, macromolecules and hormones, as well as that of platelets and blood cells. Through control of vascular tone, EC regulate the regional blood flow. They also direct inflammatory cells to foreign materials, areas in need of repair or defense against infections. In addition, EC are important in controlling blood fluidity, platelet adhesion and aggregation, leukocyte activation, adhesion, and transmigration. They also tightly keep the balance between coagulation and fibrinolysis and play a major role in the regulation of immune responses, inflammation and angiogenesis. To fulfill these different tasks, EC are heterogeneous and perform distinctly in the various organs and along the vascular tree. Important morphological, physiological and phenotypic differences between EC in the different parts of the arterial tree as well as between arteries and veins optimally support their specified functions in these vascular areas. This review updates the current knowledge about the morphology and function of endothelial cells, particularly their differences in different localizations around the body paying attention specifically to their different responses to physical, biochemical and environmental stimuli considering the different origins of the EC.

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.

Liver sinusoidal endothelial cells (LSECs) line the low shear, sinusoidal capillary channels of the liver and are the most abundant non-parenchymal hepatic cell population. LSECs do not simply form a barrier within the hepatic sinusoids but have vital physiological and immunological functions, including filtration, endocytosis, antigen presentation and leukocyte recruitment. Reflecting these multifunctional properties, LSECs display unique structural and phenotypic features that differentiate them from the capillary endothelium present within other organs. It is now clear that LSECs have a critical role in maintaining immune homeostasis within the liver and in mediating the immune response during acute and chronic liver injury. In this Review, we outline how LSECs influence the immune microenvironment within the liver and discuss their contribution to immune-mediated liver diseases and the complications of fibrosis and carcinogenesis.

Blood vessels are lined by endothelial cells engaged in distinct organ-specific functions but little is known about their characteristic gene expression profiles. RNA-Sequencing of the brain, lung, and heart endothelial translatome identified specific pathways, transporters and cell-surface markers expressed in the endothelium of each organ, which can be visualized at http://www.rehmanlab.org/ribo . We found that endothelial cells express genes typically found in the surrounding tissues such as synaptic vesicle genes in the brain endothelium and cardiac contractile genes in the heart endothelium. Complementary analysis of endothelial single cell RNA-Seq data identified the molecular signatures shared across the endothelial translatome and single cell transcriptomes. The tissue-specific heterogeneity of the endothelium is maintained during systemic in vivo inflammatory injury as evidenced by the distinct responses to inflammatory stimulation. Our study defines endothelial heterogeneity and plasticity and provides a molecular framework to understand organ-specific vascular disease mechanisms and therapeutic targeting of individual vascular beds. Blood vessels supply nutrients, oxygen and other key molecules to all of the organs in the body. Cells lining the blood vessels, called endothelial cells, regulate which molecules pass from the blood to the organs they supply. For example, brain endothelial cells prevent toxic molecules from getting into the brain, and lung endothelial cells allow immune cells into the lungs to fight off bacteria or viruses. Determining which genes are switched on in the endothelial cells of major organs might allow scientists to determine what endothelial cells do in the brain, heart, and lung, and how they differ; or help scientists deliver drugs to a particular organ. If endothelial cells from different organs switch on different groups of genes, each of these groups of genes can be thought of as a ‘genetic signature’ that identifies endothelial cells from a specific organ. Now, Jambusaria et al. show that brain, heart, and lung endothelial cells have distinct genetic signatures. The experiments used mice that had been genetically modified to have tags on their endothelial cells. These tags made it possible to isolate RNA – a molecule similar to DNA that contains the information about which genes are active – from endothelial cells without separating the cells from their tissue of origin. Next, RNA from endothelial cells in the heart, brain and lung was sequenced and analyzed. The results show that each endothelial cell type has a distinct genetic signature under normal conditions and infection-like conditions. Unexpectedly, the experiments also showed that genes that were thought to only be switched on in the cells of specific tissues are also on in the endothelial cells lining the blood vessels of the tissue. For example, genes switched on in brain cells are also active in brain endothelial cells, and genes allowing heart muscle cells to pump are also on in the endothelial cells of the heart blood vessels. The endothelial cell genetic signatures identified by Jambusaria et al. can be used as “postal codes” to target drugs to a specific organ via the endothelial cells that feed it. It might also be possible to use these genetic signatures to build organ-specific blood vessels from stem cells in the laboratory. Future work will try to answer why endothelial cells serving the heart and brain use genes from these organs.

The transcription factor FOXO1 is identified as a crucial checkpoint of vascular growth, coupling the metabolic and proliferative activities of endothelial cells. FOXO1 is a checkpoint of vascular growth The mechanisms that balance the metabolism of endothelial cells and their growth state are not known. Here Michael Potente and colleagues identify the transcription factor FOXO1 as a crucial checkpoint of vascular growth, coupling the metabolic and proliferative activities of endothelial cells. They find that FOXO1 expression in endothelial cells is required to keep the cells quiescent, through suppressing c-MYC signalling, thereby reducing glycolysis and mitochondrial respiration. Endothelial-specific deletion of FOXO1 in mice induces vessel hyperplasia and enlargement. Endothelial cells (ECs) are plastic cells that can switch between growth states with different bioenergetic and biosynthetic requirements 1 . Although quiescent in most healthy tissues, ECs divide and migrate rapidly upon proangiogenic stimulation 2 , 3 . Adjusting endothelial metabolism to the growth state is central to normal vessel growth and function 1 , 4 , yet it is poorly understood at the molecular level. Here we report that the forkhead box O (FOXO) transcription factor FOXO1 is an essential regulator of vascular growth that couples metabolic and proliferative activities in ECs. Endothelial-restricted deletion of FOXO1 in mice induces a profound increase in EC proliferation that interferes with coordinated sprouting, thereby causing hyperplasia and vessel enlargement. Conversely, forced expression of FOXO1 restricts vascular expansion and leads to vessel thinning and hypobranching. We find that FOXO1 acts as a gatekeeper of endothelial quiescence, which decelerates metabolic activity by reducing glycolysis and mitochondrial respiration. Mechanistically, FOXO1 suppresses signalling by MYC (also known as c-MYC), a powerful driver of anabolic metabolism and growth 5 , 6 . MYC ablation impairs glycolysis, mitochondrial function and proliferation of ECs while its EC-specific overexpression fuels these processes. Moreover, restoration of MYC signalling in FOXO1-overexpressing endothelium normalizes metabolic activity and branching behaviour. Our findings identify FOXO1 as a critical rheostat of vascular expansion and define the FOXO1–MYC transcriptional network as a novel metabolic checkpoint during endothelial growth and proliferation.

Cells derived from pluripotent sources in vitro must resemble those found in vivo as closely as possible at both transcriptional and functional levels in order to be a useful tool for studying diseases and developing therapeutics. Recently, differentiation of human pluripotent stem cells (hPSCs) into brain microvascular endothelial cells (ECs) with blood–brain barrier (BBB)-like properties has been reported. These cells have since been used as a robust in vitro BBB model for drug delivery and mechanistic understanding of neurological diseases. However, the precise cellular identity of these induced brain microvascular endothelial cells (iBMECs) has not been well described. Employing a comprehensive transcriptomic metaanalysis of previously published hPSC-derived cells validated by physiological assays, we demonstrate that iBMECs lack functional attributes of ECs since they are deficient in vascular lineage genes while expressing clusters of genes related to the neuroectodermal epithelial lineage (Epi-iBMEC). Overexpression of key endothelial ETS transcription factors (ETV2, ERG, and FLI1) reprograms Epi-iBMECs into authentic endothelial cells that are congruent with bona fide endothelium at both transcriptomic as well as some functional levels. This approach could eventually be used to develop a robust human BBB model in vitro that resembles the human brain EC in vivo for functional studies and drug discovery.

The bone marrow microenvironment has a key role in regulating haematopoiesis, but its molecular complexity and response to stress are incompletely understood. Here we map the transcriptional landscape of mouse bone marrow vascular, perivascular and osteoblast cell populations at single-cell resolution, both at homeostasis and under conditions of stress-induced haematopoiesis. This analysis revealed previously unappreciated levels of cellular heterogeneity within the bone marrow niche and resolved cellular sources of pro-haematopoietic growth factors, chemokines and membrane-bound ligands. Our studies demonstrate a considerable transcriptional remodelling of niche elements under stress conditions, including an adipocytic skewing of perivascular cells. Among the stress-induced changes, we observed that vascular Notch delta-like ligands (encoded by Dll1 and Dll4 ) were downregulated. In the absence of vascular Dll4 , haematopoietic stem cells prematurely induced a myeloid transcriptional program. These findings refine our understanding of the cellular architecture of the bone marrow niche, reveal a dynamic and heterogeneous molecular landscape that is highly sensitive to stress and illustrate the utility of single-cell transcriptomic data in evaluating the regulation of haematopoiesis by discrete niche populations. The transcriptional landscape of cell populations of the mouse bone marrow microenvironment, mapped at single-cell resolution, reveals cellular heterogeneity in this niche as well as substantial transcriptional remodelling under stress conditions.

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.

The high selectivity of the human blood-brain barrier (BBB) restricts delivery of many pharmaceuticals and therapeutic antibodies to the central nervous system. Here, we describe an in vitro microfluidic organ-on-a-chip BBB model lined by induced pluripotent stem cell-derived human brain microvascular endothelium interfaced with primary human brain astrocytes and pericytes that recapitulates the high level of barrier function of the in vivo human BBB for at least one week in culture. The endothelium expresses high levels of tight junction proteins and functional efflux pumps, and it displays selective transcytosis of peptides and antibodies previously observed in vivo. Increased barrier functionality was accomplished using a developmentally-inspired induction protocol that includes a period of differentiation under hypoxic conditions. This enhanced BBB Chip may therefore represent a new in vitro tool for development and validation of delivery systems that transport drugs and therapeutic antibodies across the human BBB. In vitro blood-brain barrier (BBB) models do not fully recapitulate the in vivo barrier function. Here the authors develop an organ-on-a-chip BBB model using iPS-derived human brain endothelial cells differentiated under hypoxia, primary human pericytes and astrocytes, which maintains in vivo-like BBB barrier and shuttling functions for a week.