Specialized Endothelial Cells Research Articles

A novel efficient strategy to generate liver sinusoidal endothelial cells from human pluripotent stem cells

Liver sinusoidal endothelial cells (LSECs) are highly specialized endothelial cells (ECs) that play an important role in liver development and regeneration. Additionally, it is involved in various pathological processes, including steatosis, inflammation, fibrosis and hepatocellular carcinoma. However, the rapid dedifferentiation of LSECs after culture greatly limits their use in vitro modeling for biomedical applications. In this study, we developed a highly efficient protocol to induce LSEC-like cells from human induced pluripotent stem cells (hiPSCs) in only 8 days. Using single-cell transcriptomic analysis, we identified several novel LSEC-specific markers, such as EPAS1, LIFR, and NID1, as well as several previously revealed markers, such as CLEC4M, CLEC1B, CRHBP and FCN3. These LSEC markers are specifically expressed in our LSEC-like cells. Furthermore, hiPSC-derived cells expressed LSEC-specific proteins and exhibited LSEC-related functions, such as the uptake of acetylated low density lipoprotein (ac-LDL) and immune complex endocytosis. Overall, this study confirmed that our novel protocol allowed hiPSCs to rapidly acquire an LSEC-like phenotype and function in vitro. The ability to generate LSECs efficiently and rapidly may help to more precisely mimic liver development and disease progression in a liver-specific multicellular microenvironment, offering new insights into the development of novel therapeutic strategies.

Liver sinusoidal cells eliminate blood-borne phage K1F.

Phage treatment has regained attention due to an increase in multiresistant bacteria. For phage therapy to be successful, phages must reach their target bacteria in sufficiently high numbers. Blood-borne phages are believed to be captured by macrophages in the liver and spleen. Since liver sinusoids also consist of specialized scavenger liver sinusoidal endothelial cells (LSECs) and Kupffer cells (KCs), this study investigated the contribution of both cell types in the elimination of Escherichia coli phage K1Fg10b::gfp (K1Fgfp) in mice. Circulatory half-life, organ, and hepatocellular distribution of K1Fgfp were determined following intravenous administration. Internalization of K1Fgfp and effects of phage opsonization on uptake were explored using primary mouse and human LSEC and KC cultures. When inoculated with 107 virions, >95% of the total K1Fgfp load was eliminated from the blood within 20 min, and 94% of the total retrieved K1Fgfp was localized to the liver. Higher doses resulted in slower elimination, possibly reflecting temporary saturation of liver scavenging capacity. Phage DNA was detected in both cell types, with a KC:LSEC ratio of 12:1 per population following cell isolation. Opsonization with plasma proteins increased time-dependent cellular uptake in both LSECs and KCs in vitro. Internalized phages were rapidly transported along the endocytic pathway to lysosomal compartments. Reduced viability of intracellular K1Fgfp corroborated inactivation following endocytosis. This study is the first to identify phage distribution in the liver at the hepatocellular level, confirming clearance of K1Fgfp performed mostly by KCs with a significant uptake also in LSECs.IMPORTANCEFaced with the increasing amounts of bacteria with multidrug antimicrobial resistance, phage therapy has regained attention as a possible treatment option. The phage field has recently experienced an emergence in commercial interest as research has identified new and more efficient ways of identifying and matching phages against resistant superbugs. Currently, phages are unapproved drugs in most parts of the world. For phages to reach broad clinical use, they must be shown to be clinically safe and useful. The results presented herein contribute to increased knowledge about the pharmacokinetics of the T7-like phage K1F in the mammalian system. The cell types of the liver that are responsible for rapid phage blood clearance are identified. Our results highlight the need for more research about appropriate dose regimens when phage therapy is delivered intravenously and advise essential knowledge about cell systems that should be investigated further for detailed phage pharmacodynamics.

A novel study of brain microvascular endothelial cells induced by astrocyte conditioned medium for constructing blood brain barrier model in vitro: A promising tool for meningitis of teleost

The blood-brain barrier (BBB) is mainly composed of specialized endothelial cells, which can resist harmful substances, transport nutrients, and maintain the stability of the brain environment. In this study, an endothelial cell line from tilapia (Oreochromis niloticus) named TVEC-01 was successfully established. During the earlier establishment phase of the cell line, the TVEC-01 cells were persistently exposed to an astrocyte-conditioned medium (ACM). TVEC-01 cells were identified as an endothelial cell line. TVEC-01 cells retained the multiple functions of endothelial cells and were capable of performing various experiments in vitro. Furthermore, TVEC-01 cells efficiently expressed BBB-related tight junctions and key efflux transporters. From the results of the qRT-PCR, we found that the TVEC-01 cell line did not gradually lose BBB characteristics after persistent and repetitive passages, which was different from the vast majority of immortalized endothelial cells. The results showed that ACM induced up-regulation of the expression levels of multiple BBB-related genes in TVEC-01 cells. We confirmed that Streptococcus agalactiae was capable of invading the TVEC-01 cells and initiating a series of immune responses, which provided a theoretical basis for S. agalactiae to break through the BBB of teleost through the transcellular traversal pathway. In summary, we have successfully constructed an endothelial cell line of teleost, named TVEC-01, which can be used in many experiments in vitro and even for constructing BBB in vitro. Moreover, it was confirmed that S. agalactiae broke through the BBB of teleost through the transcellular traversal pathway and caused meningitis.

Host-microbe interactions at the blood-brain barrier through the lens of induced pluripotent stem cell-derived brain-like endothelial cells.

Microbe-induced meningoencephalitis/meningitis is a life-threatening infection of the central nervous system (CNS) that occurs when pathogens are able to cross the blood-brain barrier (BBB) and gain access to the CNS. The BBB consists of highly specialized brain endothelial cells that exhibit specific properties to allow tight regulation of CNS homeostasis and prevent pathogen crossing. However, during meningoencephalitis/meningitis, the BBB fails to protect the CNS. Modeling the BBB remains a challenge due to the specialized characteristics of these cells. In this review, we cover the induced pluripotent stem cell-derived, brain-like endothelial cell model during host-pathogen interaction, highlighting the strengths and recent work on various pathogens known to interact with the BBB. As stem cell technologies are becoming more prominent, the stem cell-derived, brain-like endothelial cell model has been able to reveal new insights in vitro, which remain challenging with other in vitro cell-based models consisting of primary human brain endothelial cells and immortalized human brain endothelial cell lines.

Nanomaterials as drug delivery agents for overcoming the blood-brain barrier: A comprehensive review.

The blood-brain barrier (BBB), a critical interface of specialized endothelial cells, plays a pivotal role in regulating molecular and ion transport between the central nervous system (CNS) and systemic circulation. This review aims to delve into the intricate architecture and functions of the BBB while addressing challenges associated with delivering therapeutics to the brain. Historical milestones and contemporary insights underscore the BBB's significance in protecting the CNS. Innovative approaches for enhanced drug transport include intranasal delivery exploiting olfactory and trigeminal pathways, as well as techniques like temporary BBB opening through chemicals, receptors, or focused ultrasound. These avenues hold the potential to reshape conventional drug delivery paradigms and address the limitations posed by the BBB's selectivity. This review underscores the vital role of the BBB in maintaining CNS health and emphasizes the importance of effective drug delivery through this barrier. Nanoparticles emerge as promising candidates to overcome BBB limitations and potentially revolutionize the treatment of CNS disorders. As research progresses, the application of nanomaterials shows immense potential for advancing neurological therapeutics, albeit with careful consideration of safety aspects.

The RNA-binding protein CSDE1 promotes hematopoietic stem and progenitor cell generation via translational control of Wnt signaling.

In vertebrates, the earliest hematopoietic stem and progenitor cells (HSPCs) are derived from a subset of specialized endothelial cells, hemogenic endothelial cells, in the aorta-gonad-mesonephros region through endothelial-to-hematopoietic transition. HSPC generation is efficiently and accurately regulated by a variety of factors and signals; however, the precise control of these signals remains incompletely understood. Post-transcriptional regulation is crucial for gene expression, as the transcripts are usually bound by RNA-binding proteins (RBPs) to regulate RNA metabolism. Here, we report that the RBP protein Csde1-mediated translational control is essential for HSPC generation during zebrafish early development. Genetic mutants and morphants demonstrated that depletion of csde1 impaired HSPC production in zebrafish embryos. Mechanistically, Csde1 regulates HSPC generation through modulating Wnt/β-catenin signaling activity. We demonstrate that Csde1 binds to ctnnb1 mRNAs (encoding β-catenin, an effector of Wnt signaling) and regulates translation but not stability of ctnnb1 mRNA, which further enhances β-catenin protein level and Wnt signal transduction activities. Together, we identify Csde1 as an important post-transcriptional regulator and provide new insights into how Wnt/β-catenin signaling is precisely regulated at the post-transcriptional level.

Gold nanoparticle delivery to solid tumors: a multiparametric study on particle size and the tumor microenvironment.

Nanoparticle (NP) delivery to solid tumors remains an actively studied field, where several recent studies have shed new insights into the underlying mechanisms and the still overall poor efficacy. In the present study, Au NPs of different sizes were used as model systems to address this topic, where delivery of the systemically administered NPs to the tumor as a whole or to tumor cells specifically was examined in view of a broad range of tumor-associated parameters. Using non-invasive imaging combined with histology, immunohistochemistry, single-cell spatial RNA expression and image-based single cell cytometry revealed a size-dependent complex interaction of multiple parameters that promoted tumor and tumor-cell specific NP delivery. Interestingly, the data show that most NPs are sequestered by tumor-associated macrophages and cancer-associated fibroblasts, while only few NPs reach the actual tumor cells. While perfusion is important, leaky blood vessels were found not to promote NP delivery, but rather that delivery efficacy correlated with the maturity level of tumor-associated blood vessels. In line with recent studies, we found that the presence of specialized endothelial cells, expressing high levels of CD276 and Plvap promoted both tumor delivery and tumor cell-specific delivery of NPs. This study identifies several parameters that can be used to determine the suitability of NP delivery to the tumor region or to tumor cells specifically, and enables personalized approaches for maximal delivery of nanoformulations to the targeted tumor.

Generation & characterization of expandable human liver sinusoidal endothelial cells and their application to assess hepatotoxicity in an advanced in vitro liver model

Liver sinusoidal endothelial cells (LSECs) are highly specialized endothelial cells forming the hepatic sinusoidal wall. Besides their high endocytic potential, LSECs have been demonstrated to markedly contribute to liver homeostasis and immunity, and may partially explain unexpected hepatotoxicity of drug candidates. However, their use for in vitro investigations is compromised by poor cell yields and a limited proliferation capacity. Here, we report the transient expansion of primary human LSECs from three donors by lentiviral transduction. Transduced (“upcyte®”) LSECs were able to undergo at least 25 additional population doublings (PDs) before growth arrest due to senescence. Expanded upcyte® LSECs maintained several characteristics of primary LSECs, including expression of surface markers such as MMR and LYVE-1 as well as rapid uptake of acetylated LDL and ovalbumin. We further investigated the suitability of expanded upcyte® LSECs and proliferating upcyte® hepatocytes for detecting acetaminophen toxicity at millimolar concentrations (0, 0.5, 1, 2, 5, 10 mM) in static 2D cultures and a microphysiological 3D model. upcyte® LSECs exhibited a higher sensitivity to acetaminophen-induced toxicity compared to upcyte® hepatocytes in 2D culture, however, culturing upcyte® LSECs together with upcyte® hepatocytes in a co-culture reduced APAP-induced toxicity compared to 2D monocultures. A perfused Dynamic42 3D model was more sensitive to acetaminophen than the 2D co-culture model. Cytotoxicity in the 3D model was evident by decreased cellular viability, elevated LDH release, reduced nuclei counts and impaired cell morphology. Taken together, our data demonstrate that transient expansion of LSECs represents a suitable method for generation of large quantities of cells while maintaining many characteristics of primary cells and responsiveness to acetaminophen.

Small-world connectivity dictates collective endothelial cell signaling

Every blood vessel is lined by a single layer of highly specialized, yet adaptable and multifunctional endothelial cells. These cells, the endothelium, control vascular contractility, hemostasis, and inflammation and regulate the exchange of oxygen, nutrients, and waste products between circulating blood and tissue. To control each function, the endothelium processes endlessly arriving requests from multiple sources using separate clusters of cells specialized to detect specific stimuli. A well-developed but poorly understood communication system operates between cells to integrate multiple lines of information and coordinate endothelial responses. Here, the nature of the communication network has been addressed using single-cell Ca2+ imaging across thousands of endothelial cells in intact blood vessels. Cell activities were cross-correlated and compared to a stochastic model to determine network connections. Highly correlated Ca2+ activities occurred in scattered cell clusters, and network communication links between them exhibited unexpectedly short path lengths. The number of connections between cells (degree distribution) followed a power-law relationship revealing a scale-free network topology. The path length and degree distribution revealed an endothelial network with a “small-world” configuration. The small-world configuration confers particularly dynamic endothelial properties including high signal-propagation speed, stability, and a high degree of synchronizability. Local activation of small clusters of cells revealed that the short path lengths and rapid signal transmission were achieved by shortcuts via connecting extensions to nonlocal cells. These findings reveal that the endothelial network design is effective for local and global efficiency in the interaction of the cells and rapid and robust communication between endothelial cells in order to efficiently control cardiovascular activity.

Group B Streptococcus-Induced Macropinocytosis Contributes to Bacterial Invasion of Brain Endothelial Cells.

Bacterial meningitis is defined as serious inflammation of the central nervous system (CNS) in which bacteria infect the blood–brain barrier (BBB), a network of highly specialized brain endothelial cells (BECs). Dysfunction of the BBB is a hallmark of bacterial meningitis. Group B Streptococcus (GBS) is one of the leading organisms that cause bacterial meningitis, especially in neonates. Macropinocytosis is an actin-dependent form of endocytosis that is also tightly regulated at the BBB. Previous studies have shown that inhibition of actin-dependent processes decreases bacterial invasion, suggesting that pathogens can utilize macropinocytotic pathways for invasion. The purpose of this project is to study the factors that lead to dysfunction of the BBB. We demonstrate that infection with GBS increases rates of endocytosis in BECs. We identified a potential pathway, PLC-PKC-Nox2, in BECs that contributes to macropinocytosis regulation. Here we demonstrate that downstream inhibition of PLC, PKC, or Nox2 significantly blocks GBS invasion of BECs. Additionally, we show that pharmacological activation of PKC can turn on macropinocytosis and increase bacterial invasion of nonpathogenic yet genetically similar Lactococcus lactis. Our results suggest that GBS activates BEC signaling pathways that increase rates of macropinocytosis and subsequently the invasion of GBS.

Endothelial Cell Metabolism in Vascular Functions.

Simple SummaryRecent findings in the field of vascular biology are nourishing the idea that targeting the endothelial cell metabolism may be an alternative strategy to antiangiogenic therapy, as well as a novel therapeutic approach for cardiovascular disease. Deepening the molecular mechanisms regulating how ECs re-adapt their metabolic status in response to the changeable conditions of the tissue microenvironment may be beneficial to develop novel innovative treatments to counteract the aberrant growth of vasculature.The endothelium is the innermost layer of all blood and lymphatic vessels composed of a monolayer of specialized endothelial cells (ECs). It is regarded as a dynamic and multifunctional endocrine organ that takes part in essential processes, such as the control of blood fluidity, the modulation of vascular tone, the regulation of immune response and leukocyte trafficking into perivascular tissues, and angiogenesis. The inability of ECs to perform their normal biological functions, known as endothelial dysfunction, is multi-factorial; for instance, it implicates the failure of ECs to support the normal antithrombotic and anti-inflammatory status, resulting in the onset of unfavorable cardiovascular conditions such as atherosclerosis, coronary artery disease, hypertension, heart problems, and other vascular pathologies. Notably, it is emerging that the ability of ECs to adapt their metabolic status to persistent changes of the tissue microenvironment could be vital for the maintenance of vascular functions and to prevent adverse vascular events. The main purpose of the present article is to shed light on the unique metabolic plasticity of ECs as a prospective therapeutic target; this may lead to the development of novel strategies for cardiovascular diseases and cancer.

Blood brain barrier as an interface for alcohol induced neurotoxicity during development

Ethanol consumption during pregnancy or lactation permanently impairs the development of the central nervous system (CNS), resulting in the spectrum of fetal alcohol disorders (FASD). FASD is a general term that covers a set of deficits in the embryo caused by gestational alcohol exposure, with fetal alcohol syndrome (FAS) considered the most serious. The clinical features of FAS include facial abnormalities, short stature, low body weight, and evidence of structural and/or functional damage to the central nervous system (CNS). The prevalence of FAS carriers worldwide is about 15 for every 10,000 live births (about 119,000 children with APS born per year). Epidemiological data in the US show that the incidence of FAS exceeds other congenital syndromes such as Down syndrome and spina bifida. The deleterious effects of ethanol appear in different brain regions, varying according to the dose and period of neural development when the embryo was exposed, and include: 1) microcephaly; 2) abnormalities in cortical development, with a significant decrease in gyrification; 3) agenesis or hypoplasia of the corpus callosum; and 4) cognitive and behavioral deficits (such as impaired memory and learning, speech difficulties, and hyperactivity). Current evidence indicates that CNS blood vessels are particularly affected by teratogenic ethanol. The CNS vasculature is composed of specialized endothelial cells that establish intimate interactions with astrocytes, pericytes, and microglia, constituting the neurovascular unit of the blood-brain barrier (BBB). Together with the fact that BBB exert protective function, it can prevent the passage of substances and drugs to treat diseases that affect the CNS. Pathological changes in the BBB, such as drug abuse during pregnancy, congenital infections, or ageing processes can drastically alter the molecular structure and vascular stability, disrupting the BBB and aggravating certain neurodegenerative and neurological diseases. In this review, we address the effects of alcohol exposure on the formation of the BBB, specifically describing the cellular and molecular events induced by ethanol in the physiology of endothelial cells and glial cells, as well as their interaction during CNS development.

Induced Pluripotent Stem Cell (iPSC)-Derived Endothelial Cells to Study Bacterial-Brain Endothelial Cell Interactions.

Bacterial meningitis is a serious infection of the central nervous system (CNS) that occurs when blood-borne bacteria are able to exit the cerebral vasculature and cause inflammation. The blood-brain barrier (BBB) and the meningeal blood-CSF barrier (mBCSFB) are composed of highly specialized brain endothelial cells (BECs) that possess unique phenotypes when compared to their peripheral endothelial counterparts. To cause meningitis, bacterial pathogens must be able to interact and penetrate these specialized BECs to gain access to the CNS. In vitro models have been employed to study bacterial-BEC interactions; however, many lack BEC phenotypes. Induced pluripotent stem cell (iPSC) technologies have enabled the derivation of brain endothelial-like cells that phenocopy BECs in culture. Recently, these iPSC-BECs have been employed to examine the host-pathogen interaction at the endothelial brain barriers. Using two clinically relevant human meningeal pathogens, this chapter describes the use of iPSC-BECs to study various aspects of BEC-bacterial interaction.

3115 – THE COLLAGEN-MODIFYING LYSYL HYDROXYLASE, PLOD2, IS REQUIRED FOR HEMATOPOIETIC STEM CELL SPECIFICATION

Hematopoietic stem cells (HSCs) continuously replenish the hematopoietic system and form the basis of transplant therapies for hematologic and autoimmune diseases. In vertebrates, HSCs are developmentally specified from a transient population of specialized endothelial cells known as hemogenic endothelium (HE) located in the floor of the dorsal aorta. Under the influence of intrinsic and extrinsic differentiation cues, HE cells lose their endothelial fate and acquire hematopoietic identity through an endothelial-to-hematopoietic transition (EHT). Decades of intensive study have identified key peptide signals required for HSC specification, but a complete understanding remains elusive. Extracellular matrix (ECM) is a major component of the HSC inductive environment able both to signal directly and also regulate display of known signaling factors, but few or no studies have directly addressed the role of ECM in HSC specification. Collagen, the most abundant protein in vertebrates, forms the structural backbone of ECM and its maturation into stiffened fibers is regulated by Plod2 (Lysyl Hydroxylase 2), which catalyzes hydroxylation of key collagen lysines as a precondition to fibril crosslinking. Plod2 thus regulates ECM stability, permeability, and stiffness, and has previous roles described in malignant epithelial-to-mesenchymal transition (EMT) and metastasis. We have performed in vivo expression and functional studies in the zebrafish model. Our data show that plod2 is expressed in tissues adjacent to the dorsal aorta at the time of EHT, while plod2 loss-of-function by targeted deletion or antisense abrogates emergence of HSCs in the AGM. We are currently extending our findings to mammals. Our studies identify ECM architecture and its enzymatic regulation as central to the establishment of definitive hematopoiesis. Hematopoietic stem cells (HSCs) continuously replenish the hematopoietic system and form the basis of transplant therapies for hematologic and autoimmune diseases. In vertebrates, HSCs are developmentally specified from a transient population of specialized endothelial cells known as hemogenic endothelium (HE) located in the floor of the dorsal aorta. Under the influence of intrinsic and extrinsic differentiation cues, HE cells lose their endothelial fate and acquire hematopoietic identity through an endothelial-to-hematopoietic transition (EHT). Decades of intensive study have identified key peptide signals required for HSC specification, but a complete understanding remains elusive. Extracellular matrix (ECM) is a major component of the HSC inductive environment able both to signal directly and also regulate display of known signaling factors, but few or no studies have directly addressed the role of ECM in HSC specification. Collagen, the most abundant protein in vertebrates, forms the structural backbone of ECM and its maturation into stiffened fibers is regulated by Plod2 (Lysyl Hydroxylase 2), which catalyzes hydroxylation of key collagen lysines as a precondition to fibril crosslinking. Plod2 thus regulates ECM stability, permeability, and stiffness, and has previous roles described in malignant epithelial-to-mesenchymal transition (EMT) and metastasis. We have performed in vivo expression and functional studies in the zebrafish model. Our data show that plod2 is expressed in tissues adjacent to the dorsal aorta at the time of EHT, while plod2 loss-of-function by targeted deletion or antisense abrogates emergence of HSCs in the AGM. We are currently extending our findings to mammals. Our studies identify ECM architecture and its enzymatic regulation as central to the establishment of definitive hematopoiesis.

How Many Cell Types Are in the Kidney and What Do They Do?

The kidney maintains electrolyte, water, and acid-base balance, eliminates foreign and waste compounds, regulates blood pressure, and secretes hormones. There are at least 16 different highly specialized epithelial cell types in the mammalian kidney. The number of specialized endothelial cells, immune cells, and interstitial cell types might even be larger. The concerted interplay between different cell types is critical for kidney function. Traditionally, cells were defined by their function or microscopical morphological appearance. With the advent of new single-cell modalities such as transcriptomics, epigenetics, metabolomics, and proteomics we are entering into a new era of cell type definition. This new technological revolution provides new opportunities to classify cells in the kidney and understand their functions.

Opening the window for endothelial-to-hematopoietic transition

Definitive long-term hematopoietic stem cells (LT-HSCs) arise during embryogenesis in a process termed endothelial-to-hematopoietic transition (EHT), in which specialized hemogenic endothelial cells (HECs) transform into hematopoietic cells. The transcription factor RUNX1 marks HECs and is essential for EHT. Ectopic RUNX1 expression in non-HECs is sufficient to convert them into HECs. However, the conversion efficiency depends on the developmental timing of expression. In this issue of Genes & Development, Howell and colleagues (pp. 1475-1489) leverage this observation to further understand how RUNX1 mediates EHT. They engineered mice that ectopically express RUNX1 in endothelial cells at different developmental time points and doses. They then performed chromatin accessibility and other analyses and correlate this with hemogenic potential. They found that RUNX1 collaborates with TGFβ signaling transcription factors to drive chromatin accessibility changes that specify HECs. They also highlight interesting parallels between EHT and endothelial-to-mesenchymal transition (EndoMT), which occurs during cardiac development. The results of Howell and colleagues provide new mechanistic insights into EHT and take us one step closer to generating patient-specific LT-HSCs from induced pluripotent stem cells.

The Blood-Brain Barrier, Oxidative Stress, and Insulin Resistance.

The blood–brain barrier (BBB) is a network of specialized endothelial cells that regulates substrate entry into the central nervous system (CNS). Acting as the interface between the periphery and the CNS, the BBB must be equipped to defend against oxidative stress and other free radicals generated in the periphery to protect the CNS. There are unique features of brain endothelial cells that increase the susceptibility of these cells to oxidative stress. Insulin signaling can be impacted by varying levels of oxidative stress, with low levels of oxidative stress being necessary for signaling and higher levels being detrimental. Insulin must cross the BBB in order to access the CNS, levels of which are important in peripheral metabolism as well as cognition. Any alterations in BBB transport due to oxidative stress at the BBB could have downstream disease implications. In this review, we cover the interactions of oxidative stress at the BBB, how insulin signaling is related to oxidative stress, and the impact of the BBB in two diseases greatly affected by oxidative stress and insulin resistance: diabetes mellitus and Alzheimer’s disease.

Blood-Brain Barrier Dysfunction in CNS Disorders and Putative Therapeutic Targets: An Overview.

The blood-brain barrier (BBB) is a fundamental component of the central nervous system (CNS). Its functional and structural integrity is vital to maintain the homeostasis of the brain microenvironment by controlling the passage of substances and regulating the trafficking of immune cells between the blood and the brain. The BBB is primarily composed of highly specialized microvascular endothelial cells. These cells’ special features and physiological properties are acquired and maintained through the concerted effort of hemodynamic and cellular cues from the surrounding environment. This complex multicellular system, comprising endothelial cells, astrocytes, pericytes, and neurons, is known as the neurovascular unit (NVU). The BBB strictly controls the transport of nutrients and metabolites into brain parenchyma through a tightly regulated transport system while limiting the access of potentially harmful substances via efflux transcytosis and metabolic mechanisms. Not surprisingly, a disruption of the BBB has been associated with the onset and/or progression of major neurological disorders. Although the association between disease and BBB disruption is clear, its nature is not always evident, specifically with regard to whether an impaired BBB function results from the pathological condition or whether the BBB damage is the primary pathogenic factor prodromal to the onset of the disease. In either case, repairing the barrier could be a viable option for treating and/or reducing the effects of CNS disorders. In this review, we describe the fundamental structure and function of the BBB in both healthy and altered/diseased conditions. Additionally, we provide an overview of the potential therapeutic targets that could be leveraged to restore the integrity of the BBB concomitant to the treatment of these brain disorders.

Hlf Expression Marks Early Emergence of Hematopoietic Stem Cell Precursors With Adult Repopulating Potential and Fate.

In the aorta-gonad-mesonephros (AGM) region of mouse embryos, pre-hematopoietic stem cells (pre-HSCs) are generated from rare and specialized hemogenic endothelial cells (HECs) via endothelial-to-hematopoietic transition, followed by maturation into bona fide hematopoietic stem cells (HSCs). As HECs also generate a lot of hematopoietic progenitors not fated to HSCs, powerful tools that are pre-HSC/HSC-specific become urgently critical. Here, using the gene knockin strategy, we firstly developed an Hlf-tdTomato reporter mouse model and detected Hlf-tdTomato expression exclusively in the hematopoietic cells including part of the immunophenotypic CD45– and CD45+ pre-HSCs in the embryonic day (E) 10.5 AGM region. By in vitro co-culture together with long-term transplantation assay stringent for HSC precursor identification, we further revealed that unlike the CD45– counterpart in which both Hlf-tdTomato-positive and negative sub-populations harbored HSC competence, the CD45+ E10.5 pre-HSCs existed exclusively in Hlf-tdTomato-positive cells. The result indicates that the cells should gain the expression of Hlf prior to or together with CD45 to give rise to functional HSCs. Furthermore, we constructed a novel Hlf-CreER mouse model and performed time-restricted genetic lineage tracing by a single dose induction at E9.5. We observed the labeling in E11.5 AGM precursors and their contribution to the immunophenotypic HSCs in fetal liver (FL). Importantly, these Hlf-labeled early cells contributed to and retained the size of the HSC pool in the bone marrow (BM), which continuously differentiated to maintain a balanced and long-term multi-lineage hematopoiesis in the adult. Therefore, we provided another valuable mouse model to specifically trace the fate of emerging HSCs during development.

Protein Kinase C-Dependent Von Willebrand Factor Secretion Is a Dual-Phased Process That Originates from 2 Distinct Intracellular Compartments

Introductionvon Willebrand factor (VWF) and VWF-propeptide (VWFpp) are stored for secretion within specialized endothelial cell secretory organelles called Weibel-Palade bodies (WPBs). Protein kinase C (PKC) is a key signaling intermediary for a number of endothelial agonists that trigger the release of VWF, such as Vascular Endothelial Growth Factor (VEGF) and Shiga toxin Stx1B. While both agonists elevate intracellular free Ca2+ ([Ca2+]i) and simultaneously either signal through the novel, Ca2+-independent PKCδ (VEGF) or utilize the classical, Ca2+-dependent PKCα (Stx1B), there has been speculation whether an additional effector pathway may be operating during PKC-mediated VWF secretion. Phorbol 12-myristate 13-acetate (PMA), an activator of both classical and novel protein kinase C (PKC) isoforms, is also a potent VWF secretagogue, and it is generally assumed that PMA-evoked VWF release arises from exocytosis of WPBs.ResultsHere we present evidence that PMA elicits a dual-phased secretory response with an early (0-15 minutes) and a late (>15 minutes) stage. A large fraction of the early phase of PMA-evoked VWF and VWFpp secretion arises from a cycloheximide (CHX)-sensitive (i.e., nascent or non-storage) compartment, and can be blocked by pre-treatment with the broad spectrum PKC inhibitor bisindolylmaleimide I (BIS I). Histamine-induced VWFpp secretion during the early phase is insensitive to CHX or BIS I. VEGF induced a sharp rise of VWFpp secretion in the early phase and a much slower increase in the later phase. The early phase was also substantially reduced by CHX and BIS I, suggesting release from the same non-storage pool that is mobilized by PMA.High speed imaging of WPB exocytosis and [Ca2+]i in cells expressing VWFpp-EGFP targeted to WPBs revealed that the majority of PMA- and VEGF-induced VWFpp secretion during the early phase did not originate from WPBs. Histamine stimulation was associated with a sharp rise in [Ca2+]i, followed by rapid exocytosis within seconds of 41.4±4.0% (mean±sem, n=12 cells, 322 fusion events) of fluorescent WPBs. PMA did not elevate [Ca2+]i, led to a slow gradual increase in fusion events after a long and highly variable delay (357±63 seconds) and resulted in release of 12.4±3.3% (n=13 cells, 133 fusion events) of VWFpp-EGFP labeled WPBs. VEGF induced a complex [Ca2+]i increase, and after considerable delay (37.8 ±5.9 seconds) this was followed by WPB release of 18.2±3.5% (n=14 cells, 181 fusion events) of fluorescent WPBs. Because all three stimuli release quantitatively similar amounts of VWFpp during the early phase, the discrepancy in the fractions of WPBs undergoing degranulation suggests that PKC signaling mobilizes VWFpp from a compartment other than WPBs.Analysis of PMA-evoked VWFpp secretion showed that pre-treatment with the potent phospholipase D1/2 inhibitor 5-fluoro-2-indolyl des-chlorohalopemide (FIPI) significantly reduced the early, CHX-sensitive component of PMA-evoked VWFpp secretion, which suggests that this pool originates from the trans -Golgi network. Silencing of syntaxin-6, which we identify as a novel immature WPB-SNARE, and the recycling endosome regulator Rab11 revealed that PMA-evoked VWFpp secretion does not traverse through a non-classical post-Golgi compartment such as the recycling endosome on its way out.During the late phase PMA leads to almost complete degranulation of WPBs, but the mechanism has so far remained elusive. Time-lapse imaging of PMA-, histamine- and VEGF-stimulated WPB degranulation showed that this was insensitive to CHX treatment. In support of a role in sensitizing WPB release towards [Ca2+]i we found that a short pre-treatment with PMA enhanced the ability of ionomycin to induce Ca2+-driven VWFpp secretion at sub-maximal but not maximal doses. PMA-potentiated, ionomycin-induced VWFpp secretion was insensitive to CHX, underscoring that this originated from WPBs.ConclusionsOur data show that two pathways operate for rapid regulated secretion of VWF, one involving WPB exocytosis and the other, regulated by PKC activation, that accesses VWF and VWFpp in a late biosynthetic compartment, most likely the trans -Golgi network. Our findings also have significant implications for the mechanistic interpretation of data obtained from biochemical studies of WPB exocytosis induced by PMA, where secreted VWF has been assumed to arise solely from WPBs. DisclosuresNo relevant conflicts of interest to declare.