A brain-specific angiogenic mechanism enabled by tip cell specialization.

Vertebrate organs require locally adapted blood vessels1,2. The gain of such organotypic vessel specializations is often deemed to be molecularly unrelated to the process of organ vascularization. Here, opposing this model, we reveal a molecular mechanism for brain-specific angiogenesis that operates under the control of Wnt7a/b ligands-well-known blood-brain barrier maturation signals3-5. The control mechanism relies on Wnt7a/b-dependent expression of Mmp25, which we find is enriched in brain endothelial cells. CRISPR-Cas9 mutagenesis in zebrafish reveals that this poorly characterized glycosylphosphatidylinositol-anchored matrix metalloproteinase is selectively required in endothelial tip cells to enable their initial migration across the pial basement membrane lining the brain surface. Mechanistically, Mmp25 confers brain invasive competence by cleaving meningeal fibroblast-derived collagen IV α5/6 chains within a short non-collagenous region of the central helical part of the heterotrimer. After genetic interference with the pial basement membrane composition, the Wnt-ß-catenin-dependent organotypic control of brain angiogenesis is lost, resulting in properly patterned, yet blood-brain-barrier-defective cerebrovasculatures. We reveal an organ-specific angiogenesis mechanism, shed light on tip cell mechanistic angiodiversity and thereby illustrate how organs, by imposing local constraints on angiogenic tip cells, can select vessels matching their distinctive physiological requirements.

Effects of intermittent hypoxia with thrombin in an in vitro model of human brain endothelial cells and their impact on PAR-1/PAR-3 cleavage.

Patients with obstructive sleep apnea/hypopnea (OSA) are at high risk of cerebrovascular diseases leading to cognitive impairment. The oxidative stress generated by intermittent hypoxia (IH) could lead to an increase in blood-brain barrier (BBB) permeability, an essential interface for the protection of the brain. Moreover, in patients with OSA, blood coagulation could be increased leading to cardiovascular complications. Thrombin is a factor found increased in these populations that exerts various cellular effects through activation of protease activated receptors (PARs). Thus, we have evaluated in an in vitro BBB model the association of IH with thrombin at two concentrations. We measured the apparent BBB permeability, expression of tight junctions, ROS production, HIF-1α expression, and cleavage of PAR-1/PAR-3. Pre-treatment with dabigatran was performed. IH and higher thrombin concentrations altered BBB permeability: high levels of HIF-1α expression, ROS and PAR-1 activation compared to PAR-3 in such conditions. Conversely, lower concentration of thrombin associated with IH appear to have a protective effect on BBB with a significant cleavage of PAR-3. Dabigatran reversed the deleterious effect of thrombin at high concentrations but also suppressed the beneficial effect of low dose thrombin. Therefore, thrombin and PARs represent novel attractive targets to prevent BBB opening in OSA.

Murine Beta-Amyloid (1-42) Oligomers Disrupt Endothelial Barrier Integrity and VEGFR Signaling via Activating Astrocytes to Release Deleterious Soluble Factors.

Transgenic mouse models of Alzheimer's disease (AD) overexpress mutations of the human amyloid protein precursor (APP) and presenilin-1 (PSEN1) genes, which are known causes of amyloid pathology in familial AD. However, animal models for studying AD in the context of aging and age-related co-morbidities, such as blood-brain barrier (BBB) disruptions, are lacking. More recently, aged and progeroid mouse models have been proposed as alternatives to study aging-related AD, but the toxicity of murine amyloid-beta protein (Aß) is not well defined. In this study, we aimed to study the potential toxicity of murine Aß on brain endothelial cells and astrocytes, which are important components of the BBB, using mouse brain endothelial cells (bEnd.3) and astrocytes (C8-D1A). Murine-soluble Aß (1-42) oligomers (sAßO42) (10 µM) induced negligible injuries in an endothelial monolayer but induced significant barrier disruptions in a bEnd.3 and C8-D1A co-culture. Similar results of endothelial perturbation were observed in a bEnd.3 monolayer treated with astrocyte-conditioned medium (ACM) generated by astrocytes exposed to sAßO42 (ACM-sAßO42), while additional exogenous sAßO42 did not cause further damage. Western blot analysis showed that ACM-sAßO42 altered the basal activities of vascular endothelial growth factor receptor 2 (VEGFR2), eNOS, and the signaling of the MEK/ERK and Akt pathways in bEnd.3. Our results showed that murine sAßO42 was moderately toxic to an endothelial and astrocyte co-culture. These damaging effects on the endothelial barrier were induced by deleterious soluble factors released from astrocytes, which disrupted endothelial VEGFR2 signaling and perturbed cell survival and barrier stabilization.

Engineered human blood-brain barrier microfluidic model for vascular permeability analyses.

The blood-brain barrier (BBB) greatly restricts the entry of biological and engineered therapeutic molecules into the brain. Due to challenges in translating results from animal models to the clinic, relevant in vitro human BBB models are needed to assess pathophysiological molecular transport mechanisms and enable the design of targeted therapies for neurological disorders. This protocol describes an in vitro model of the human BBB self-assembled within microfluidic devices from stem-cell-derived or primary brain endothelial cells, and primary brain pericytes and astrocytes. This protocol requires 1.5 d for device fabrication, 7 d for device culture and up to 5 d for downstream imaging, protein and gene expression analyses. Methodologies to measure the permeability of any molecule in the BBB model, which take 30 min per device, are also included. Compared with standard 2D assays, the BBB model features relevant cellular organization and morphological characteristics, as well as values of molecular permeability within the range expected in vivo. These properties, coupled with a functional brain endothelial expression profile and the capability to easily test several repeats with low reagent consumption, make this BBB model highly suitable for widespread use in academic and industrial laboratories.

Embryonic Pericytes Promote Microglial Homeostasis and Their Effects on Neural Progenitors in the Developing Cerebral Cortex.

Multifaceted microglial functions in the developing brain, such as promoting the differentiation of neural progenitors and contributing to the positioning and survival of neurons, have been progressively revealed. Although previous studies have noted the relationship between vascular endothelial cells and microglia in the developing brain, little attention has been given to the importance of pericytes, the mural cells surrounding endothelial cells. In this study, we attempted to dissect the role of pericytes in microglial distribution and function in developing mouse brains. Our immunohistochemical analysis showed that approximately half of the microglia attached to capillaries in the cerebral walls. Notably, a magnified observation of the position of microglia, vascular endothelial cells and pericytes demonstrated that microglia were preferentially associated with pericytes that covered 79.8% of the total capillary surface area. Through in vivo pericyte depletion induced by the intraventricular administration of a neutralizing antibody against platelet-derived growth factor receptor (PDGFR)ß (clone APB5), we found that microglial density was markedly decreased compared with that in control antibody-treated brains because of their low proliferative capacity. Moreover, in vitro coculture of isolated CD11b+ microglia and NG2+PDGFRα- cells, which are mostly composed of pericytes, from parenchymal cells indicated that pericytes promote microglial proliferation via the production of soluble factors. Furthermore, pericyte depletion by APB5 treatment resulted in a failure of microglia to promote the differentiation of neural stem cells into intermediate progenitors. Taken together, our findings suggest that pericytes facilitate microglial homeostasis in the developing brains, thereby indirectly supporting microglial effects on neural progenitors.SIGNIFICANCE STATEMENT This study highlights the novel effect of pericytes on microglia in the developing mouse brain. Through multiple analyses using an in vivo pericyte depletion mouse model and an in vitro coculture study of isolated pericytes and microglia from parenchymal cells, we demonstrated that pericytes contribute to microglial proliferation and support microglia in efficiently promoting the differentiation of neural stem cells into intermediate progenitors. Our present data provide evidence that pericytes function not only in the maintenance of cerebral microcirculation and blood brain barrier (BBB) integrity but also in microglial homeostasis in the developing cerebral walls. These findings will expand our knowledge and help elucidate the mechanism of brain development both in healthy and disease conditions.

Role of the Extracellular Traps in Central Nervous System.

It has been reported that several immune cells can release chromatin and granular proteins into extracellular space in response to the stimulation, forming extracellular traps (ETs). The cells involved in the extracellular trap formation are recognized including neutropils, macrophages, basophils, eosinophils, and mast cells. With the development of research related to central nervous system, the role of ETs has been valued in neuroinflammation, blood-brain barrier, and other fields. Meanwhile, it has been found that microglial cells as the resident immune cells of the central nervous system can also release ETs, updating the original understanding. This review aims to clarify the role of the ETs in the central nervous system, especially in neuroinflammation and blood-brain barrier.

Nano-scale architecture of blood-brain barrier tight-junctions.

Tight junctions (TJs) between blood-brain barrier (BBB) endothelial cells construct a robust physical barrier, whose damage underlies BBB dysfunctions related to several neurodegenerative diseases. What makes these highly specialized BBB-TJs extremely restrictive remains unknown. Here, we use super-resolution microscopy (dSTORM) to uncover new structural and functional properties of BBB TJs. Focusing on three major components, Nano-scale resolution revealed sparse (occludin) vs. clustered (ZO1/claudin-5) molecular architecture. In mouse development, permeable TJs become first restrictive to large molecules, and only later to small molecules, with claudin-5 proteins arrangement compacting during this maturation process. Mechanistically, we reveal that ZO1 clustering is independent of claudin-5 in vivo. In contrast to accepted knowledge, we found that in the developmental context, total levels of claudin-5 inversely correlate with TJ functionality. Our super-resolution studies provide a unique perspective of BBB TJs and open new directions for understanding TJ functionality in biological barriers, ultimately enabling restoration in disease or modulation for drug delivery.

Pericytes as mediators of infiltration of macrophages in multiple sclerosis.

BACKGROUND: Multiple sclerosis (MS) is a neurodegenerative condition of the central nervous system (CNS). It is associated with blood-brain barrier (BBB) breakdown and intravasation of leukocytes, particularly monocyte-derived macrophages, into the CNS. Pericytes are mural cells that are encased within the basement membrane of vasculature, and they contribute functionally to the neurovascular unit. These cells play an important role in maintaining BBB integrity and CNS homeostasis. However, the critical role of pericytes in mediating inflammation in MS or its models is unclear. Whether pericytes infiltrate into the CNS parenchyma in MS also needs clarification. METHODS: CNS samples from the experimental autoimmune encephalomyelitis (EAE) mouse model of MS were collected at different time points for immunohistochemical analysis of pericytes along the inflamed vasculature. These findings were validated using MS brain specimens, and further analysis of pericyte involvement in inflammation was carried out by culturing primary pericytes and macrophages. Multiplex ELISA, transmigration assay and real-time PCR were used to study the inflammatory potential of pericytes in cultures. RESULTS: We found that pericytes exhibit a heterogenous morphology, with notable elongation in the inflamed perivascular cuffs of EAE. This was manifested by a decrease in pericyte density but an increase in the coverage by pericytes along the vasculature. Chondroitin sulfate proteoglycans (CSPGs), a family of extracellular matrix proteins enriched within inflamed perivascular cuffs, elevated levels of pro-inflammatory chemokines/cytokines in pericytes in culture. Importantly, pericytes stimulated with CSPGs enhanced macrophage migration. We did not detect pericytes in the CNS parenchyma during EAE, and this was corroborated in MS brain samples. CONCLUSIONS: Our data suggest that pericytes seek to restore the BBB through increased coverage, but that their exposure to CSPGs prompt their facilitation of macrophages to enter the CNS to elevate neuroinflammation in EAE and MS.

Recent progress and new challenges in modeling of human pluripotent stem cell-derived blood-brain barrier.

The blood-brain barrier (BBB) is a semipermeable unit that serves to vascularize the central nervous system (CNS) while tightly regulating the movement of molecules, ions, and cells between the blood and the brain. The BBB precisely controls brain homeostasis and protects the neural tissue from toxins and pathogens. The BBB is coordinated by a tight monolayer of brain microvascular endothelial cells, which is subsequently supported by mural cells, astrocytes, and surrounding neuronal cells that regulate the barrier function with a series of specialized properties. Dysfunction of barrier properties is an important pathological feature in the progression of various neurological diseases. In vitro BBB models recapitulating the physiological and diseased states are important tools to understand the pathological mechanism and to serve as a platform to screen potential drugs. Recent advances in this field have stemmed from the use of pluripotent stem cells (PSCs). Various cell types of the BBB such as brain microvascular endothelial cells (BMECs), pericytes, and astrocytes have been derived from PSCs and synergistically incorporated to model the complex BBB structure in vitro. In this review, we summarize the most recent protocols and techniques for the differentiation of major cell types of the BBB. We also discuss the progress of BBB modeling by using PSC-derived cells and perspectives on how to reproduce more natural BBBs in vitro.

Influence of basal media composition on barrier fidelity within human pluripotent stem cell-derived blood-brain barrier models.

It is increasingly recognized that brain microvascular endothelial cells (BMECs), the principal component of the blood-brain barrier (BBB), are highly sensitive to soluble cues from both the bloodstream and the brain. This concept extends in vitro, where the extracellular milieu can also influence BBB properties in cultured cells. However, the extent to which baseline culture conditions can affect BBB properties in vitro remains unclear, which has implications for model variability and reproducibility, as well as downstream assessments of molecular transport and disease phenotypes. Here, we explore this concept by examining BBB properties within human-induced pluripotent stem cell (iPSC)-derived BMEC-like cells cultured under serum-free conditions in DMEM/F12 and Neurobasal media, which have fully defined compositions. We demonstrate notable differences in both passive and active BBB properties as a function of basal media composition. Further, RNA sequencing and phosphoproteome analyses revealed alterations to various signaling pathways in response to basal media differences. Overall, our results demonstrate that baseline culture conditions can have a profound influence on the performance of in vitro BBB models, and these effects should be considered when designing experiments that utilize such models for basic research and preclinical assays.

The protective effects of Pimavanserin against cerebral ischemia-induced brain injury.

The integrity of the blood-brain barrier (BBB) is mainly maintained by the brain vascular endothelial cells and the tight junctions amongst them. Pimavanserin is a novel agent approved for the treatment of Parkinson's disease and exerts neuroprotective properties. The present study aims to explore the possibility that Pimavanserin might be an effective agent used for the treatment of cerebral ischemia stroke. Middle cerebral artery occlusion (MCAO) was established in mice, and oxygen-glucose deprivation/reoxygenation (OGD/R) was established in brain bEND.3 endothelial cells. Mice were randomly divided into four groups: (1) Sham operation group; (2). Pimavanserin (1 mg/kg); (3). MCAO; (4). Pimavanserin+ MCAO. We found that compared to the Sham group, the elevated neurological deficit score and brain water content increased production of inflammatory factors, increased BBB permeability, and downregulated Claudin 5 expression were observed in the MCAO group and were all dramatically reversed by the administration of Pimavanserin. Brain bEND.3 endothelial cells were treated with Pimavanserin before the exposure to OGD/R. The significantly increased lactate dehydrogenase (LDH) release, declined cell viability, increased endothelial permeability, downregulated Claudin 5 and Krüppel-like factors 6 (KLF6) were observed in the OGD/R group and were all reversed by the introduction of Pimavanserin. Lastly, the effects of Pimavanserin on the expression level of Claudin 5 and endothelial permeability in OGD/R-challenged endothelial cells were both abolished by the knockdown of KLF6. Taken together, our data revealed that Pimavanserin protected against cerebral ischemia injury by regulating the BBB integrity in a KLF6-dependent manner.

Semimechanistic Population Pharmacokinetic Modeling to Investigate Amyloid Beta Trafficking and Accumulation at the BBB Endothelium.

Elevated exposure to toxic amyloid beta (Aß) peptides and consequent blood-brain barrier (BBB) dysfunction are believed to promote vasculopathy in Alzheimer's disease (AD). However, the accumulation kinetics of different Aß isoforms within the BBB endothelium and how it drives BBB dysfunction are not clearly characterized. Using single positron emission computed tomography (SPECT)-computed tomography (CT) dynamic imaging coupled with population pharmacokinetic modeling, we investigated the accumulation kinetics of Aß40 and Aß42 in the BBB endothelium. Brain clearance was quantified after intracerebral administration of 125I-Aß, and BBB-mediated transport was shown to account for 54% of 125I-Aß40 total clearance. A brain influx study demonstrated lower values of both maximal rate (Vmax) and Michaelis constant (Km) for 125I-Aß42 compared to 125I-Aß40. Validated by a transcytosis study in polarized human BBB endothelial cell (hCMEC/D3) monolayers, model simulations demonstrated impaired exocytosis was responsible for inefficient permeability and enhanced accumulation of Aß42 in the BBB endothelium. Further, both isoforms were shown to disrupt the exocytosis machinery of BBB endothelial cells so that a vicious cycle could be generated. The validated model was able to capture changes in Aß steady-state levels in plasma as well as the brain during AD progression and allowed us to predict the kinetics of Aß accumulation in the BBB endothelium.

A Proton-Coupled Transport System for ß-Hydroxy-ß-Methylbutyrate (HMB) in Blood-Brain Barrier Endothelial Cell Line hCMEC/D3.

ß-Hydroxy-ß-methylbutyrate (HMB), a leucine metabolite, is used as a nutritional ingredient to improve skeletal muscle health. Preclinical studies indicate that this supplement also elicits significant benefits in the brain; it promotes neurite outgrowth and prevents age-related reductions in neuronal dendrites and cognitive performance. As orally administered HMB elicits these effects in the brain, we infer that HMB crosses the blood-brain barrier (BBB). However, there have been no reports detailing the transport mechanism for HMB in BBB. Here we show that HMB is taken up in the human BBB endothelial cell line hCMEC/D3 via H+-coupled monocarboxylate transporters that also transport lactate and ß-hydroxybutyrate. MCT1 (monocarboxylate transporter 1) and MCT4 (monocarboxylate transporter 4) belonging to the solute carrier gene family SLC16 (solute carrier, gene family 16) are involved, but additional transporters also contribute to the process. HMB uptake in BBB endothelial cells results in intracellular acidification, demonstrating cotransport with H+. Since HMB is known to activate mTOR with potential to elicit transcriptomic changes, we examined the influence of HMB on the expression of selective transporters. We found no change in MCT1 and MCT4 expression. Interestingly, the expression of LAT1 (system L amino acid transporter 1), a high-affinity transporter for branched-chain amino acids relevant to neurological disorders such as autism, is induced. This effect is dependent on mTOR (mechanistic target of rapamycine) activation by HMB with no involvement of histone deacetylases. These studies show that HMB in systemic circulation can cross the BBB via carrier-mediated processes, and that it also has a positive influence on the expression of LAT1, an important amino acid transporter in the BBB.

Application and advantages of zebrafish model in the study of neurovascular unit.

The concept of "Neurovascular Unit" (NVU) was put forward, so that the research goal of Central Nervous System (CNS) diseases gradually transitioned from a single neuron to the structural and functional integrity of the NVU. Zebrafish has the advantages of high homology with human genes, strong reproductive capacity and visualization of neural circuits, so it has become an emerging model organism for NVU research and has been applied to a variety of CNS diseases. Based on CNKI (https://www.cnki.net/) and PubMed (https://pubmed.ncbi.nlm.nih.gov/about/) databases, the author of this article sorted out the relevant literature, analyzed the construction of a zebrafish model of various CNS diseases，and the use of diagrams showed the application of zebrafish in the NVU, revealed its relationship, which would provide new methods and references for the treatment and research of CNS diseases.

Investigating receptor-mediated antibody transcytosis using blood-brain barrier organoid arrays.

BACKGROUND: The pathways that control protein transport across the blood-brain barrier (BBB) remain poorly characterized. Despite great advances in recapitulating the human BBB in vitro, current models are not suitable for systematic analysis of the molecular mechanisms of antibody transport. The gaps in our mechanistic understanding of antibody transcytosis hinder new therapeutic delivery strategy development. METHODS: We applied a novel bioengineering approach to generate human BBB organoids by the self-assembly of astrocytes, pericytes and brain endothelial cells with unprecedented throughput and reproducibility using micro patterned hydrogels. We designed a semi-automated and scalable imaging assay to measure receptor-mediated transcytosis of antibodies. Finally, we developed a workflow to use CRISPR/Cas9 gene editing in BBB organoid arrays to knock out regulators of endocytosis specifically in brain endothelial cells in order to dissect the molecular mechanisms of receptor-mediated transcytosis. RESULTS: BBB organoid arrays allowed the simultaneous growth of more than 3000 homogenous organoids per individual experiment in a highly reproducible manner. BBB organoid arrays showed low permeability to macromolecules and prevented transport of human non-targeting antibodies. In contrast, a monovalent antibody targeting the human transferrin receptor underwent dose- and time-dependent transcytosis in organoids. Using CRISPR/Cas9 gene editing in BBB organoid arrays, we showed that clathrin, but not caveolin, is required for transferrin receptor-dependent transcytosis. CONCLUSIONS: Human BBB organoid arrays are a robust high-throughput platform that can be used to discover new mechanisms of receptor-mediated antibody transcytosis. The implementation of this platform during early stages of drug discovery can accelerate the development of new brain delivery technologies.

Role of TRP ion channels in cerebral circulation and neurovascular communication.

The dynamic regulation of blood flow is essential for meeting the high metabolic demands of the brain and maintaining brain function. Cerebral blood flow is regulated primarily by 1) the intrinsic mechanisms that determine vascular contractility and 2) signals from neurons and astrocytes that alter vascular contractility. Stimuli from neurons and astrocytes can also initiate a signaling cascade in the brain capillary endothelium to increase regional blood flow. Recent studies provide evidence that TRP channels in endothelial cells, smooth muscle cells, neurons, astrocytes, and perivascular nerves control cerebrovascular contractility and cerebral blood flow. TRP channels exert their functional effects either through cell membrane depolarization or by serving as a Ca2+ influx pathway. Endothelial cells and astrocytes also maintain the integrity of the blood-brain barrier. Both endothelial cells and astrocytes express TRP channels, and an increase in endothelial TRP channel activity has been linked with a disrupted endothelial barrier function. Therefore, TRP channels can play a potentially important role in regulating blood-brain barrier integrity. Here, we review the regulation of cerebrovascular contractility by TRP channels under healthy and disease conditions and their potential roles in maintaining blood-brain barrier function.

Human Blood Vessel Organoids Penetrate Human Cerebral Organoids and Form a Vessel-Like System.

Vascularization of tissues, organoids and organ-on-chip models has been attempted using endothelial cells. However, the cultured endothelial cells lack the capacity to interact with other somatic cell types, which is distinct from developing vascular cells in vivo. Recently, it was demonstrated that blood vessel organoids (BVOs) recreate the structure and functions of developing human blood vessels. However, the tissue-specific adaptability of BVOs had not been assessed in somatic tissues. Herein, we investigated whether BVOs infiltrate human cerebral organoids and form a blood-brain barrier. As a result, vascular cells arising from BVOs penetrated the cerebral organoids and developed a vessel-like architecture composed of CD31+ endothelial tubes coated with SMA+ or PDGFR+ mural cells. Molecular markers of the blood-brain barrier were detected in the vascularized cerebral organoids. We revealed that BVOs can form neural-specific blood-vessel networks that can be maintained for over 50 days.

CCR6 Expression on B Cells Is Not Required for Clinical or Pathological Presentation of MOG Protein-Induced Experimental Autoimmune Encephalomyelitis despite an Altered Germinal Center Response.

B cells have been implicated in the pathogenesis of multiple sclerosis, but the mechanisms that guide B cell activation in the periphery and subsequent migration to the CNS remain incompletely understood. We previously showed that systemic inflammation induces an accumulation of B cells in the spleen in a CCR6/CCL20-dependent manner. In this study, we evaluated the role of CCR6/CCL20 in the context of myelin oligodendrocyte glycoprotein (MOG) protein-induced (B cell-dependent) experimental autoimmune encephalomyelitis (EAE). We found that CCR6 is upregulated on murine B cells that migrate into the CNS during neuroinflammation. In addition, human B cells that migrate across CNS endothelium in vitro were found to be CCR6+, and we detected CCL20 production by activated CNS-derived human endothelial cells as well as a systemic increase in CCL20 protein during EAE. Although mice that lack CCR6 expression specifically on B cells exhibited an altered germinal center reaction in response to MOG protein immunization, CCR6-deficient B cells did not exhibit any competitive disadvantage in their migration to the CNS during EAE, and the clinical and pathological presentation of EAE induced by MOG protein was unaffected. These data, to our knowledge, provide new information on the role of B cell-intrinsic CCR6 expression in a B cell-dependent model of neuroinflammation.

Azilsartan protects against hyperglycemia-induced hyperpermeability of the blood-brain barrier.

Diabetes mellitus (DM) is a complex metabolic disease with significant neurological complications and is reported to be closely related to the blood-brain barrier (BBB) disruption. Azilsartan is an antagonist of the Angiotensin II receptor developed for the treatment of hypertension, and it has been recently reported to have neuroprotective effects. The present study aims to investigate the protective effect of Azilsartan against hyperglycemia-induced BBB disruption and its underlying mechanism. Male db/db mice were treated with Azilsartan (20 µg/day) for 10 consecutive days. Compared to the control group, increased BBB permeability, suppressed occludin expression, excessive release of inflammatory factors, and downregulation of krüppel-like factor 2 (KLF2) were observed in diabetic mice, all of which were dramatically reversed by Azilsartan treatment. In the in vitro experiments, elevated endothelial permeability and decreased expression of occludin and KLF2 were observed in high glucose-challenged endothelial cells, which were significantly alleviated by Azilsartan. Lastly, the silencing of KLF2 abolished the protective effects of Azilsartan against the high glucose-induced expression of occludin and endothelial monolayer permeability in bEnd.3 brain endothelial cells. Based on these observations, we concluded that Azilsartan protected against hyperglycemia-induced hyperpermeability of BBB via the KLF2/occludin axis.

Blood-Brain Barrier and Neurodegenerative Diseases-Modeling with iPSC-Derived Brain Cells.

The blood-brain barrier (BBB) regulates the delivery of oxygen and important nutrients to the brain through active and passive transport and prevents neurotoxins from entering the brain. It also has a clearance function and removes carbon dioxide and toxic metabolites from the central nervous system (CNS). Several drugs are unable to cross the BBB and enter the CNS, adding complexity to drug screens targeting brain disorders. A well-functioning BBB is essential for maintaining healthy brain tissue, and a malfunction of the BBB, linked to its permeability, results in toxins and immune cells entering the CNS. This impairment is associated with a variety of neurological diseases, including Alzheimer's disease and Parkinson's disease. Here, we summarize current knowledge about the BBB in neurodegenerative diseases. Furthermore, we focus on recent progress of using human-induced pluripotent stem cell (iPSC)-derived models to study the BBB. We review the potential of novel stem cell-based platforms in modeling the BBB and address advances and key challenges of using stem cell technology in modeling the human BBB. Finally, we highlight future directions in this area.