The blood-brain barrier.

The blood–brain barrier is playing a critical role in controlling the influx and efflux of biological substances essential for the brain’s metabolic activity as well as neuronal function. Thus, the functional and structural integrity of the BBB is pivotal to maintain the homeostasis of the brain microenvironment. The different cells and structures contributing to developing this barrier are summarized along with the different functions that BBB plays at the brain–blood interface. We also explained the role of shear stress in maintaining BBB integrity. Furthermore, we elaborated on the clinical aspects that correlate between BBB disruption and different neurological and pathological conditions. Blood vessels are critical to deliver oxygen and nutrients to all of the tissues and organs throughout the body. The blood vessels that vascularize the central nervous system (CNS) possess unique properties, termed the blood–brain barrier, which allow these vessels to tightly regulate the movement of ions, molecules, and cells between the blood and the brain. This precise control of CNS homeostasis allows for proper neuronal function and also protects the neural tissue from toxins and pathogens, and alterations of these barrier properties are an important component of pathology and progression of different neurological diseases. The physiological barrier is coordinated by a series of physical, transport, and metabolic properties possessed by the endothelial cells (ECs) that form the walls of the blood vessels, and these properties are regulated by interactions with different vascular, immune, and neural cells. Understanding how these different cell populations interact to regulate the barrier properties is essential for understanding how the brain functions during health and disease.

Blood vessels are critical to deliver oxygen and nutrients to tissues and organs throughout the body. The blood vessels that vascularize the central nervous system (CNS) possess unique properties, termed the blood-brain barrier (BBB), which allow these vessels to tightly regulate the movement of ions, molecules, and cells between the blood and the brain. This precise control of CNS homeostasis allows for proper neuronal function and protects the neural tissue from toxins and pathogens, and alterations of this barrier are important components of the pathogenesis and progression of various neurological diseases. The physiological barrier is coordinated by a series of physical, transport, and metabolic properties possessed by the brain endothelial cells (ECs) that form the walls of the blood vessels. These properties are regulated by interactions between different vascular, perivascular, immune, and neural cells. Understanding how these cell populations interact to regulate barrier properties is essential for understanding how the brain functions in both health and disease contexts.

Testing of a hypothesis for osmotic opening of the blood-brain barrier.

AT THE END OF THE CENTURY before the last, a young graduate student performed an experiment that would forever change the way we look at the microvasculature of the brain. He injected basic dyes into the blood and noted that nearly every tissue of the body was colored by the dye except for the central nervous system (CNS). This experiment and the observation that bile salts could cause seizures when injected into the brain but not intravenously were the seminal 19th century observations giving rise to the concept of a blood-brain barrier (BBB). The young graduate student, Paul Ehrlich, would get many things right in his career, winning a Nobel prize in 1908 for his work on antibiotics. But the BBB he got wrong. Ehrlich thought the brain did not stain because the dye was not taken up by brain tissue, not because a barrier prevented the dye from reaching the brain. Indeed, that a BBB really existed and that it resided at the level of the capillary bed and the choroid plexus were not finally established until the late 1960s. Elegant physiological experiments by Davson and Segal (10) and electron microscopy studies by Reese and Karnovsky (24) demonstrated the basis of the BBB: the capillary bed of adult mammals is modified to exclude the production of a plasma ultrafiltrate. The major modifications include a greatly decreased rate of pinocytosis, a lack of intracellular pores and fenestrae, and obliteration of the intercellular space between brain endothelial cells by tight junctions that essentially “cement” apposing endothelial cells together. The lack of production of an ultrafiltrate means that circulating proteins such as albumin do not exude from blood into brain. This is the basis for Ehrlich’s 120-year-old observation: basic dyes bind to albumin so tightly that they are a visible proxy for plasma

The blood-brain barrier (BBB) and its selective transport mechanisms contribute to the brain homeostasis by preventing harmful substances from entering the brain and by tightly regulating nutrients exchange between the blood and the brain. The integrity and functions of the BBB are impaired during progression of various neurological diseases. As a central part in neurological diseases, BBB becomes the target during the development of brain-targeted therapies. Drugs can be designed to either directly target the BBB to restore its barrier functions and properties, or to bypass the BBB for treating diseased brain cells. In-vitro BBB models are important tools for fundamental brain research, disease modeling, and drug screening. Currently, there are unsolved problems in the conventional in-vitro BBB models. For example, the simplicity of transwell models fails to truly reproduce the complex pathophysiological environment of the BBB. The aim of this thesis is to generate an advanced BBB model that recapitulates key structures and functions of the in-vivo BBB, and could potentially improve BBB disease modeling and drug screening outcomes over the use of the transwell BBB models. In chapter 2, we generated two BBB-targeting nanobodies (Nbs) that bind to human heparin-binding EGF-like growth factor (HB-EGF). Using a transwell-based BBB model, we demonstrated that the HB-EGF targeting Nbs are able to cross the brain endothelial cells via receptor-mediated transcytosis (RMT). These Nbs could be further developed into BBB shuttle carriers for delivering therapeutical molecules into the brain. In chapter 3, we generated a BBB-on-a-chip model in a microfluidic chip platform. Our primary goal in this model was to create an in-vivo like BBB architecture. The brain endothelial cells formed a tight tube structure in the chip which can be then perfused with cell medium. The supporting neural cells (e.g., astrocytes and neurons) grew next to the endothelial tubes in a collagen-Matrigel double-ECM structure. Over time, the neural cells formed extensive networks and neural-vascular interactions. By using the HB-EGF targeting Nbs, it is shown that the microfluidic BBB chip model has better fidelity in revealing true BBB endocytosis and transcytosis efficiency of these Nbs than the transwell BBB model. The use of non-human derived cells for BBB modeling has difficulties in faithfully recapitulating human BBB physiology. In chapter 2, we differentiated hiPSCs into brain endothelial cells and used these cells to construct an induced BBB model in the transwell system. In chapter 4, we differentiated hiPSCs into functional neurons and astrocytes. We created an isogenic neural model by co-culturing these hiPSC-neural cells in the transwell system. We show that a co-culture environment is necessary in maturing both cell types. Besides, we demonstrate that the neuron-astrocyte co-culture model is suitable for modeling astrocyte-driven neuroinflammation. Bioprinting technology represents a promising approach in generating tissue-like components within a microenvironment that mimics biochemical and biophysical parameters of the brain. In chapter 5, we provide a review of neural bioprinting, which has the potential for advanced BBB engineering incorporating complex vascularized BBB structures. In chapter 6, the main findings of the thesis are summarized and discussed.

Homeostatic control of the surrounding microenvironment is a requirement for proper neuronal function. Their high metabolism requires an efficient supply of nutrients (sugars, amino acids, nucleosides, etc.) and removal of potentially toxic metabolites. A central role in balancing these two processes belongs to the barriers of the central nervous system (CNS). The Blood Brain barrier (BBB) is a dynamic interface between the peripheral circulation and the CNS that tightly regulates transport of compounds into and out of the brain. According to the currently accepted hypothesis the BBB plays a significant role in the production of the brain interstitial fluid (ISF) that directly surrounds neurons and other brain cells. However, since the ISF is only ~ 20% of the total brain volume and is distributed throughout the brain, it is difficult to sample unlike the cerebrospinal fluid (CSF). Therefore, rather less is known about its content. Amino acids (AA) are vitally necessary compounds with multiple functions. Additionally, in the CNS AAs are neurotransmitters or neurotransmitter precursors. For example, glutamine (Gln) serves as a precursor for both the major excitatory (glutamate) and inhibitory (γ-aminobutyric acid (GABA)) neurotransmitters and as a result levels are altered during some pathologies. The conditionally essential AA Gln is the most abundant AA in plasma and CSF. Moreover, in CSF Gln concentration is ~ 80% of that in plasma, while all other standard AAs are ~ 10-fold less concentrated [1]. Nevertheless, data about concentrations of Gln and other AAs in brain ISF are infrequent and inconsistent. The following Gln transporters: LAT1 (SLC7A5), SNAT2 (SLC38A2), SNAT3 (SLC38A3), and SNAT5 (SLC38A5), are significantly expressed in the BBB [2]. Moreover, LAT1 and SNAT3 have been shown to be localized on both luminal and abluminal membranes of BBB endothelial cells [3]. For my dissertation I focused on the brain ISF AAs (in particular Gln) and possible mechanisms of regulation by BBB expressed amino acid transporters (AAT)s. Using in vivo intracranial microdialysis in awake freely moving mice we determined the levels of 14 standard and 2 non-standard AA. For all AAs a ~10 fold concentration gradient between ISF and CSF was observed. Furthermore the acute administration of valine by intraperitoneal (IP) injection caused a significant increase of its concentration not only in plasma, but also in the brain ISF. In contrast, 15N2 Gln (hGln) IP administration resulted in an elevation only in plasma, but not ISF. Nonetheless this labeled form of Gln represented ~ 4% of total Gln in microdialysate samples. Thus, we demonstrated efficient transendothelial transport of both AA from the blood. Competitive inhibition of system L and system A AATs in the brain by inhibitors (2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH) and α-(methylamino)-isobutyric acid (MeAIB), respectively) introduced via the microdialysis probe led to Gln increase in the brain ISF. The Gln elevation produced due to BCH infusion was twice higher than that caused by MeAIB. To study whether Gln was released from brain cells or its raise in ISF was due to increased BBB transport, IP injection of 15N2 Gln was combined with BCH administration. The data showed a significant increase of hGln in the brain ISF in presence of BCH in comparison to its IP administration alone indicating that brain perfusion with BCH increased Gln influx via the BBB, presumably by transstimulating Gln uptake via obligatory exchanger LAT1. Since a high level of Snat5 mRNA was found in BBB its localization in brain endothelial cells (BEC) membranes was studied. Unfortunately, an antibody-dependent approach did not allow distinction of SNAT5 in the BBB endothelium. Therefore, first of all, a steep AA concentration gradient between the brain ISF and CSF was demonstrated. Second, the data are consistent with LAT1 playing a major role in the regulation of Gln in brain ISF. Since LAT1 is an obligatory exchanger, we suggest it acts together with BBB expressed SNAT3 as key regulators of Gln in brain ISF. We consider that studies using inducible endothelial specific SNAT3 knockout animals will clarify the contributions of BBB expressed SNAT3 in Gln ISF homeostasis.

The extravasation of lymphocytes across central nervous system (CNS) vascular endothelium is a key step in inflammatory demyelinating diseases including multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). The glycosaminoglycan hyaluronan (HA) and its receptor, CD44, have been implicated in this process but their precise roles are unclear. We find that CD44(-/-) mice have a delayed onset of EAE compared with wild type animals. Using an in vitro lymphocyte rolling assay, we find that fewer slow rolling (<1 μm/s) wild type (WT) activated lymphocytes interact with CD44(-/-) brain vascular endothelial cells (ECs) than with WT ECs. We also find that CD44(-/-) ECs fail to anchor HA to their surfaces, and that slow rolling lymphocyte interactions with WT ECs are inhibited when the ECs are treated with a pegylated form of the PH20 hyaluronidase (PEG-PH20). Subcutaneous injection of PEG-PH20 delays the onset of EAE symptoms by ~1 day and transiently ameliorates symptoms for 2 days following disease onset. These improved symptoms correspond histologically to degradation of HA in the lumen of CNS blood vessels, decreased demyelination, and impaired CD4(+) T-cell extravasation. Collectively these data suggest that HA tethered to CD44 on CNS ECs is critical for the extravasation of activated T cells into the CNS providing new insight into the mechanisms promoting inflammatory demyelinating disease.

Blood-Brain Barrier Breakdown and Blood-Brain Communication in Neurological and Psychiatric Diseases

The adult quiescent blood–brain barrier (BBB), a structure organised by endothelial cells through interactions with pericytes, astrocytes, neurons and microglia in the neurovascular unit, is highly regulated but fragile at the same time. In the past decade, there has been considerable progress in understanding not only the molecular pathways involved in BBB development, but also BBB breakdown in neurological diseases. Specifically, the Wnt/β-catenin, retinoic acid and sonic hedgehog pathways moved into the focus of BBB research. Moreover, angiopoietin/Tie2 signalling that is linked to angiogenic processes has gained attention in the BBB field. Blood vessels play an essential role in initiation and progression of many diseases, including inflammation outside the central nervous system (CNS). Therefore, the potential influence of CNS blood vessels in neurological diseases associated with BBB alterations or neuroinflammation has become a major focus of current research to understand their contribution to pathogenesis. Moreover, the BBB remains a major obstacle to pharmaceutical intervention in the CNS. The complications may either be expressed by inadequate therapeutic delivery like in brain tumours, or by poor delivery of the drug across the BBB and ineffective bioavailability. In this review, we initially describe the cellular and molecular components that contribute to the steady state of the healthy BBB. We then discuss BBB alterations in ischaemic stroke, primary and metastatic brain tumour, chronic inflammation and Alzheimer’s disease. Throughout the review, we highlight common mechanisms of BBB abnormalities among these diseases, in particular the contribution of neuroinflammation to BBB dysfunction and disease progression, and emphasise unique aspects of BBB alteration in certain diseases such as brain tumours. Moreover, this review highlights novel strategies to monitor BBB function by non-invasive imaging techniques focussing on ischaemic stroke, as well as novel ways to modulate BBB permeability and function to promote treatment of brain tumours, inflammation and Alzheimer’s disease. In conclusion, a deep understanding of signals that maintain the healthy BBB and promote fluctuations in BBB permeability in disease states will be key to elucidate disease mechanisms and to identify potential targets for diagnostics and therapeutic modulation of the BBB.

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Vascular endothelial cells in the central nervous system (CNS) form a barrier that restricts the movement of molecules and ions between the blood and the brain. This blood‐brain barrier (BBB) is crucial to ensure proper neuronal function and protect the CNS from injury and disease. Although the properties of the BBB are manifested in the endothelial cells, transplantation studies have demonstrated that the BBB is not intrinsic to the endothelial cells, but is induced by interactions with the neural cells. Here we use a genomic, genetic and molecular approach to elucidate the cellular and molecular mechanisms that regulate the formation of the BBB. We have identified a critical role for pericytes in regulating the permeability of CNS vessels by inhibiting the properties that make endothelial cells leaky. In particular pericytes limit the rate of transcytosis through endothelial cells as well as the expression of leukocyte adhesion molecules in CNS endothelial cells, which limits CNS immune infiltration. Furthermore, we have developed methods to highly purify and gene profile endothelial cells from different tissues, and by comparing the transcriptional profile of brain endothelial cells with those purified from the liver and lung, we have generated a comprehensive resource of transcripts that are specific to the BBB forming endothelial cells of the brain. We have further examined the profile of CNS endothelial cells following injury and disease and have identified molecular mechanisms by which pericytes control BBB formation, which are then disrupted during neurological disease leading to BBB dysfunction.

The blood-brain barrier (BBB) is essential for creating and maintaining tissue homeostasis in the central nervous system (CNS), which is key for proper neuronal function. In most vertebrates, the BBB is localized to microvascular endothelial cells that acquire barrier properties during angiogenesis of the neuroectoderm. Complex and continuous tight junctions, and the lack of fenestrae combined with low pinocytotic activity render the BBB endothelium a tight barrier for water-soluble molecules that may only enter the CNS via specific transporters. The differentiation of these unique endothelial properties during embryonic development is initiated by endothelial-specific flavours of the Wnt/β-catenin pathway in a precise spatiotemporal manner. In this review, we summarize the currently known cellular (neural precursor and endothelial cells) and molecular (VEGF and Wnt/β-catenin) mechanisms mediating brain angiogenesis and barrier formation. Moreover, we introduce more recently discovered crosstalk with cellular and acellular elements within the developing CNS such as the extracellular matrix. We discuss recent insights into the downstream molecular mechanisms of Wnt/β-catenin in particular, the recently identified target genes like Foxf2, Foxl2, Foxq1, Lef1, Ppard, Zfp551, Zic3, Sox17, Apcdd1 and Fgfbp1 that are involved in refining and maintaining barrier characteristics in the mature BBB endothelium. Additionally, we elute to recent insight into barrier heterogeneity and differential endothelial barrier properties within the CNS, focussing on the circumventricular organs as well as on the neurogenic niches in the subventricular zone and the hippocampus. Finally, open questions and future BBB research directions are highlighted in the context of taking benefit from understanding BBB development for strategies to modulate BBB function under pathological conditions.

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Ketone Bodies and Brain Metabolism: New Insights and Perspectives for Neurological Diseases.

Molecular and Magnetic Resonance Imaging of Human Embryonic Stem Cell–Derived Neural Stem Cell Grafts in Ischemic Rat Brain