Brain Capillary Endothelial Cells Research Articles

Regulatory Role of eIF2αK4 in Amino Acid Transporter Expression in Mouse Brain Capillary Endothelial Cells

Regulatory Role of eIF2αK4 in Amino Acid Transporter Expression in Mouse Brain Capillary Endothelial Cells

Activation of glial cells induces proinflammatory properties in brain capillary endothelial cells in vitro

Neurodegenerative diseases are often accompanied by neuroinflammation and impairment of the blood-brain barrier (BBB) mediated by activated glial cells through their release of proinflammatory molecules. To study the effects of glial cells on mouse brain endothelial cells (mBECs), we developed an in vitro BBB model with inflammation by preactivating mixed glial cells (MGCs) with lipopolysaccharide (LPS) before co-culturing with mBECs to study the influence of molecules released by activated MGCs. The response of the mBECs to activated MGCs was compared to direct stimulation with LPS. The cytokine profile of activated MGCs was analyzed together with their effects on the mBEC’s integrity, expression of tight junction proteins, adhesion molecules, and BBB-specific transport proteins. Stimulation of MGCs significantly upregulated mRNA expression and secretion of several pro-inflammatory cytokines. Co-culturing mBECs with pre-stimulated MGCs significantly affected the barrier integrity of mBECs similar to direct stimulation with LPS. The gene expression levels of tight junction proteins were unaltered, but tight junction proteins revealed rearrangements with respect to subcellular distribution. Compared to direct stimulation with LPS, the expression of cell-adhesion molecules was significantly increased when mBECs were co-cultured with prestimulated MGCs and thus pre-activating MGCs transforms mBECs into a proinflammatory phenotype.

Zinc-modulated bidirectional copper transfer across the blood-brain barrier in a porcine brain capillary endothelial cell culture model system

The blood-brain barrier (BBB) serves as a crucial interface, regulating the transfer of trace elements (TEs) such as copper (Cu) and zinc (Zn) between the bloodstream and the brain. Cu and Zn are essential for maintaining neural function and enzymatic processes. Understanding the interplay of Cu and Zn with the BBB is crucial for elucidating their roles in neurological health and disease. This study investigates the bidirectional transfer of Cu across the BBB and examines the impact of Zn supplementation on this process using a porcine brain capillary endothelial cell (PBCEC) model. Transendothelial electrical resistance (TEER) and capacitance measurements confirmed barrier integrity upon TE exposure, while quantification of Cu and Zn concentrations via inductively coupled plasma-tandem mass spectrometry (ICP-MS/MS) in the culture medium provided essential baseline data. Transfer studies revealed significant increases in basolateral (brain side) Cu concentrations after apical (blood side) Cu incubation, with additional Zn supplementation reducing Cu transfer from apical to basolateral compartments. Conversely, Zn incubation showed no effect on basolateral-to-apical Cu transfer. Surprisingly, it was found that Cu also transferred significantly to the apical compartments when incubated basolaterally, and with slightly higher permeability coefficients than vice versa, indicating a potential role of PBCECs in regulating Cu transport both from blood to brain and from brain to blood. These findings suggest a bidirectional Cu trafficking across PBCECs, only slightly influenced unidirectionally by Zn supplementation, highlighting the intricate interplay between TEs at the BBB. Importantly, no alterations in barrier integrity were observed, underscoring the physiological relevance of the experimental conditions. Overall, this study sheds light on the complex dynamics of Cu and Zn transfer at the BBB, emphasizing the need for comprehensive investigations into TE interactions for a deeper understanding of brain TE homeostasis.

Upregulation of Transferrin Receptor 1 (TfR1) but Not Glucose Transporter 1 (GLUT1) or CD98hc at the Blood-Brain Barrier in Response to Valproic Acid.

Transferrin receptor 1 (TfR1), glucose transporter 1 (GLUT1), and CD98hc are candidates for targeted therapy at the blood-brain barrier (BBB). Our objective was to challenge the expression of TfR1, GLUT1, and CD98hc in brain capillaries using the histone deacetylase inhibitor (HDACi) valproic acid (VPA). Primary mouse brain capillary endothelial cells (BCECs) and brain capillaries isolated from mice injected intraperitoneally with VPA were examined using RT-qPCR and ELISA. Targeting to the BBB was performed by injecting monoclonal anti-TfR1 (Ri7217)-conjugated gold nanoparticles measured using ICP-MS. In BCECs co-cultured with glial cells, Tfrc mRNA expression was significantly higher after 6 h VPA, returning to baseline after 24 h. In vivo Glut1 mRNA expression was significantly higher in males, but not females, receiving VPA, whereas Cd98hc mRNA expression was unaffected by VPA. TfR1 increased significantly in vivo after VPA, whereas GLUT1 and CD98hc were unchanged. The uptake of anti-TfR1-conjugated nanoparticles was unaltered by VPA despite upregulated TfR expression. VPA upregulates TfR1 in brain endothelium in vivo and in vitro. VPA does not increase GLUT1 and CD98hc proteins. The increase in TfR1 does not result in higher anti-TfR1 antibody targetability, suggesting targeting sufficiently occurs with available transferrin receptors without further contribution from accessory VPA-induced TfR1.

3,3',5-Triiodothyroacetic Acid Transporters.

Introduction: Thyroid hormone transporters are essential for thyroid hormones to enter target cells. Monocarboxylate transporter (MCT) 8 is a key transporter and is expressed at the blood-brain barrier (BBB), in neural cells and many other tissues. Patients with MCT8 deficiency have severe neurodevelopmental delays because of cerebral hypothyroidism and chronic sequelae of peripheral thyrotoxicosis. The T3 analog 3,3',5-triiodothyroacetic acid (TRIAC) rescued neurodevelopmental features in animal models mimicking MCT8 deficiency and improved key metabolic features in patients with MCT8 deficiency. However, the identity of the transporter(s) that facilitate TRIAC transport are unknown. Here, we screened candidate transporters that are expressed at the human BBB and/or brain-cerebrospinal fluid barrier and known thyroid hormone transporters for TRIAC transport. Materials and Methods: Plasma membrane expression was determined by cell surface biotinylation assays. Intracellular accumulation of 1 nM TRIAC was assessed in COS-1 cells expressing candidate transporters in Dulbecco's phosphate-buffered saline (DPBS)/0.1% glucose or Dulbecco's modified Eagle's medium (DMEM) with or without 0.1% bovine serum albumin (BSA). Expression of Slc22a8 was determined by fluorescent in situ hybridization in brain sections from wild-type and Mct8/Oatp1c1 knockout mice at postnatal days 12, 21, and 120. Results: In total, 59 plasma membrane transporters were selected for screening of TRIAC accumulation (n = 40 based on expression at the human BBB and/or brain-cerebrospinal fluid barrier and having small organic molecules as substrates; n = 19 known thyroid hormone transporters). Screening of the selected transporter panel showed that 18 transporters facilitated significant intracellular accumulation of TRIAC in DPBS/0.1% glucose or DMEM in the absence of BSA. In the presence of BSA, substantial transport was noted for SLCO1B1 and SLC22A8 (in DPBS/0.1% glucose and DMEM) and SLC10A1, SLC22A6, and SLC22A24 (in DMEM). The zebrafish and mouse orthologs of these transporters similarly facilitated intracellular accumulation of TRIAC. Highest Slc22a8 mRNA expression was detected in mouse brain capillary endothelial cells and choroid plexus epithelial cells at early postnatal time points, but was reduced at P120. Conclusions: Human SLC10A1, SLCO1B1, SLC22A6, SLC22A8, and SLC22A24 as well as their mouse and zebrafish orthologs are efficient TRIAC transporters. These findings contribute to the understanding of TRIAC treatment in patients with MCT8 deficiency and animal models thereof.

A face-to-face comparison of the BBB cell models hCMEC/D3 and hBMEC for their applicability to adenoviral expression of transporters.

The blood-brain barrier (BBB) is a structure mainly formed by brain capillary endothelial cells (BCEC) whose role is to regulate the exchange of compounds between the blood and the brain. In this process efflux and uptake transporters play a key role. Aim of this study was to compare the two previously established cell lines hCMEC/D3 and hBMEC as BBB cell models for the application of an adenoviral system to transiently express OATP2B1 and Pgp. Comparison of hCMEC/D3 and hBMEC mRNA and protein levels of BBB markers showed a unique expression pattern for each cell line. While showing similar expression of the efflux transporter BCRP, transferrin receptor (TFRC) and of the tight junctions proteins Occludin and ZO-1, hCMEC/D3 displayed higher levels of the endothelial marker PECAM1, VE-cadherin, Von Willebrand Factor (VWF) and of the efflux transporter Pgp. Moreover, measuring integrity of the monolayer by determining the Trans-Endothelial Electrical Resistance (TEER), electrical capacitance (CCl), and inulin apparent permeability coefficient (Papp) revealed higher TEER and lower CCl for hBMEC but comparable Papp in the two cell lines. Following adenoviral infection, enhanced OATP2B1 and Pgp expression and functionality could be observed only in hBMEC. Importantly, the adenoviral expression system did not affect expression of BBB markers and permeability in both cell lines. Taken together, our results provide first evidence that hBMEC is an applicable human BBB cell model in which adenoviral infection can be used to transiently express and investigate transporters of interest.

The putative proton-coupled organic cation antiporter is involved in uptake of triptans into human brain capillary endothelial cells

BackgroundTriptans are anti-migraine drugs with a potential central site of action. However, it is not known to what extent triptans cross the blood–brain barrier (BBB). The aim of this study was therefore to determine if triptans pass the brain capillary endothelium and investigate the possible underlying mechanisms with focus on the involvement of the putative proton-coupled organic cation (H+/OC) antiporter. Additionally, we evaluated whether triptans interacted with the efflux transporter, P-glycoprotein (P-gp).MethodsWe investigated the cellular uptake characteristics of the prototypical H+/OC antiporter substrates, pyrilamine and oxycodone, and seven different triptans in the human brain microvascular endothelial cell line, hCMEC/D3. Triptan interactions with P-gp were studied using the IPEC-J2 MDR1 cell line. Lastly, in vivo neuropharmacokinetic assessment of the unbound brain-to-plasma disposition of eletriptan was conducted in wild type and mdr1a/1b knockout mice.ResultsWe demonstrated that most triptans were able to inhibit uptake of the H+/OC antiporter substrate, pyrilamine, with eletriptan emerging as the strongest inhibitor. Eletriptan, almotriptan, and sumatriptan exhibited a pH-dependent uptake into hCMEC/D3 cells. Eletriptan demonstrated saturable uptake kinetics with an apparent Km of 89 ± 38 µM and a Jmax of 2.2 ± 0.7 nmol·min−1·mg protein−1 (n = 3). Bidirectional transport experiments across IPEC-J2 MDR1 monolayers showed that eletriptan is transported by P-gp, thus indicating that eletriptan is both a substrate of the H+/OC antiporter and P-gp. This was further confirmed in vivo, where the unbound brain-to-unbound plasma concentration ratio (Kp,uu) was 0.04 in wild type mice while the ratio rose to 1.32 in mdr1a/1b knockout mice.ConclusionsWe have demonstrated that the triptan family of compounds possesses affinity for the H+/OC antiporter proposing that the putative H+/OC antiporter plays a role in the BBB transport of triptans, particularly eletriptan. Our in vivo studies indicate that eletriptan is subjected to simultaneous brain uptake and efflux, possibly facilitated by the putative H+/OC antiporter and P-gp, respectively. Our findings offer novel insights into the potential central site of action involved in migraine treatment with triptans and highlight the significance of potential transporter related drug-drug interactions.Graphical

Molecular Characterization of the Ca2+ Release Activated Ca2+ Current in Native Brain Capillary Endothelial Cells

Brain capillary endothelial cells (cECs) form the blood-brain barrier orchestrate on-demand increases in blood flow to serve the brain’s unique metabolic needs. An in vivo imaging study showed that brain capillaries generate an array of dynamic Ca2+ signals primarily dependent on the release of Ca2+ from the endoplasmic reticulum (ER). Little is known about the physiological functions and origins of these signals. Store-operated Ca2+ entry (SOCE) is a fundamental Ca2+ influx mechanism activated by emptying ER Ca2+ stores. Following store depletion, interactions between the ER Ca2+-sensor stromal interaction molecule 1 (STIM1) and Orai1, 2, and/or 3 ion channel subunits on the plasma membrane initiate the Ca2+-release activated Ca2+ current (ICRAC) and SOCE. ICRAC and SOCE in brain cECs are uncharacterized. Therefore, we investigated STIM and Orai expression and the biophysical properties of ICRAC in primary brain cECs. Using droplet digital PCR, we found that native brain cECs express Stim1, Stim2, Orai1, and Orai3 but not Orai2. Conventional whole-cell patch-clamp electrophysiology was used to record ICRAC from freshly isolated brain cECs. Currents were induced by including the slow Ca2+ buffer EGTA in the intracellular solution. In contrast to the strong inward rectification of canonical ICRAC, currents recorded from native brain cECs were dually rectifying. The inward and outward components were blocked by La3+, enhanced with 2-APB, and inhibited with the selective Orai1 channel antagonist Synta66. Both ICRAC components were increased when a divalent-free solution (Ca2+ replaced with Cs+) was used. To gain insight into the molecular composition of CRAC channels in brain cECs, we generated EC-specific knockout (ecKO) Stim1, Orai1, and Orai3 mice. ICRAC was absent in cECs obtained from Stim1-ecKO mice. In contrast, ICRAC recorded from Orai1-ecKO mice displayed strong inward rectification with no outward current and was insensitive to Synta66. ICRAC recorded from Orai3-ecKO mice were also inwardly rectifying but were highly sensitive to Synta66. ICRAC was undetectable in cells from Orai1-Orai3-ecKO mice. These data suggest that native ICRAC in brain cECs requires the expression of Stim1, Orai1, and Orai3. The outward component of native ICRAC in these cells requires the expression of Orai1 and Orai3, but the expression of either Orai1 or Orai3 alone is suffcient to maintain the inward component. R35HL155008, R33NS115132, and P20GM130459 (to SE). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Store-operated calcium entry is necessary for sustained K+-induced capillary-to-arteriole dilation in the brain

Neurovascular coupling (NVC) is a physiological process that directs blood flow to the most active regions of the brain. The extensive network of capillaries in the brain acts as a sensory web that detects local neuronal activity and orchestrates the dilation of upstream arterioles to meet the brain’s metabolic demands. Activation of inwardly rectifying K+ (KIR) channels on brain capillary endothelial cells (cECs) by modest concentrations of extracellular K+ initiates retrograde propagating hyperpolarizing signals that induce vasodilation and increase blood flow. Brain capillaries exhibit diverse and dynamic patterns of Ca2+ signaling in vivo, but little is known about their origins or physiological function. Store-operated Ca2+ entry (SOCE) is fundamental to Ca2+homeostasis in non-excitable cells, but the contribution of SOCE to capillary-to-arteriole dilation has not been explored. Here, we report that brain cECs express the endoplasmic reticulum-resident Ca2+ sensing protein stromal interaction molecule 1 ( Stim1) and the Ca2+-selective Orai1 and Orai3 channels. Ca2+ imaging experiments using brain cECs from transgenic mice expressing GCaMP8 in the endothelium or loaded with Fluo-4 showed that these cells exhibited robust SOCE that was blocked by the Orai1 inhibitor Synta66. Using EC-specific knockout (ecKO) mice, we found that Stim1 and Orai1 but not Orai3 are required for SOCE. Ex vivo brain microvascular preparations consisting of cannulated and pressured parenchymal arterioles with intact capillary beds were used to examine the role of SOCE in capillary-to-arteriole dilation. Arteriole dilation in response to a train of K+ pulses applied to capillaries exhibited dramatic run-down when preparations were treated with Snyta66 or when tissues from Stim1-ecKO mice were used. Preparations from Orai3-ecKO mice did not demonstrate run-down. Moreover, inhibition of Ca2+-activated K+ (SK and IK) channels also induced run-down, suggesting that SOCE activates SK and IK channels to boost KIR-induced hyperpolarizing signals to dilate upstream arterioles during multiple stimulations. We conclude that brain cECs exhibit SOCE that requires Stim1 and Orai1. Our data further show that brain cEC SOCE is necessary for capillary-to-arteriole dilation during repeated stimulations, suggesting an essential role for SOCE in NVC. R35HL155008, R33NS115132, and P20GM130459 (to SE). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

A Role for iNOS in Erastin Mediated Reduction of P-Glycoprotein Transport Activity.

The blood-brain barrier is composed of both a physical barrier and an enzymatic barrier. Tight junction (TJ) proteins expressed between endothelial cells of brain capillaries provide the physical barrier to paracellular movement of ions and molecules to the brain, while luminal-facing efflux transporters enzymatically restrict the entry of blood-borne molecules from entering the brain. The expression and activity of ATP Binding Cassette transporters or "ABC" transporters in endothelial cells of the BBB and in human tumor cells are dynamically regulated by numerous signaling pathways. P-glycoprotein (P-gp), (ABCB1), is arguably the most studied transporter of the BBB, and in human cell lines. P-glycoprotein transport activity is rapidly inhibited by signaling pathways that call for the rapid production of nitric oxide (NO) from the inducible nitric oxide synthase enzyme, iNOS. This study investigated how nano-molar levels of the selective chemotherapeutic erastin affect the activity or expression of P-glycoprotein transporter in brain capillaries and in human tumor cell lines. We chose erastin because it signals to iNOS for NO production at low concentrations. Furthermore, erastin inhibits the cellular uptake of cystine through the XC- cystine/glutamate antiporter. Since previous reports indicate that NO production from iNOS can rapidly inhibit P-gp activity in tumor cells, we wondered if induction of iNOS by erastin could also rapidly reduce P-glycoprotein transport activity in brain endothelial cells and in human tumor cell lines. We show here that low concentrations of erastin (1 nM) can induce iNOS, inhibit the activity of P-glycoprotein, and reduce the intracellular uptake of cystine via the Xc- cystine/glutamate antiporter. Consistent with reduced P-glycoprotein activity in rat brain capillary endothelial cells, we show that human tumor cell lines exposed to erastin become more sensitive to cytotoxic substrates of P-glycoprotein.

Evaluation of the impact of iPSC differentiation protocols on transcriptomic signatures

Human induced pluripotent stem cells (iPSC) have the potential to produce desired target cell types in vitro and allow for the high-throughput screening of drugs/chemicals at population level thereby minimising the cost of drug discovery and drug withdrawals after clinical trials. There is a substantial need for the characterisation of the iPSC derived models to better understand and utilise them for toxicological relevant applications. In our study, iPSC (SBAD2 or SBAD3 lines obtained from StemBANCC project) were differentiated towards toxicologically relevant cell types: alveolar macrophages, brain capillary endothelial cells, brain cells, endothelial cells, hepatocytes, lung airway epithelium, monocytes, podocytes and renal proximal tubular cells. A targeted transcriptomic approach was employed to understand the effects of differentiation protocols on these cell types. Pearson correlation and principal component analysis (PCA) separated most of the intended target cell types and undifferentiated iPSC models as distinct groups with a high correlation among replicates from the same model. Based on PCA, the intended target cell types could also be separated into the three germ layer groups (ectoderm, endoderm and mesoderm). Differential expression analysis (DESeq2) presented the upregulated genes in each intended target cell types that allowed the evaluation of the differentiation to certain degree and the selection of key differentiation markers. In conclusion, these data confirm the versatile use of iPSC differentiated cell types as standardizable and relevant model systems for in vitro toxicology.

Comparison of Extracellular Vesicles from Induced Pluripotent Stem Cell-Derived Brain Cells.

The pathophysiology of many neuropsychiatric disorders is still poorly understood. Identification of biomarkers for these diseases could benefit patients due to better classification and stratification. Exosomes excreted into the circulatory system can cross the blood-brain barrier and carry a cell type-specific set of molecules. Thus, exosomes are a source of potential biomarkers for many diseases, including neuropsychiatric disorders. Here, we investigated exosomal proteins produced from human-induced pluripotent stem cells (iPSCs) and iPSC-derived neural stem cells, neural progenitors, neurons, astrocytes, microglia-like cells, and brain capillary endothelial cells. Of the 31 exosome surface markers analyzed, a subset of biomarkers were significantly enriched in astrocytes (CD29, CD44, and CD49e), microglia-like cells (CD44), and neural stem cells (SSEA4). To identify molecular fingerprints associated with disease, circulating exosomes derived from healthy control (HC) individuals were compared against schizophrenia (SCZ) patients and late-onset Alzheimer's disease (LOAD) patients. A significant epitope pattern was identified for LOAD (CD1c and CD2) but not for SCZ compared to HC. Thus, analysis of cell type- and disease-specific exosome signatures of iPSC-derived cell cultures may provide a valuable model system to explore proteomic biomarkers for the identification of novel disease profiles.

Glioma-targeted oxaliplatin/ferritin clathrate reversing the immunosuppressive microenvironment through hijacking Fe2+ and boosting Fenton reaction

Glioma is easy to develop resistance to temozolomide (TMZ). TMZ-resistant glioma secretes interleukin-10 (IL-10) and transforming growth factor-β (TGF-β), recruiting regulatory T cell (Treg) and inhibiting the activity of T cells and natural killer cell (NK cell), subsequently forming an immunosuppressive microenvironment. Oxaliplatin (OXA) greatly inhibits the proliferation of TMZ-resistant glioma cells, but the ability of OXA to cross blood–brain barrier (BBB) is weak. Thus, the therapeutic effect of OXA on glioma is not satisfactory. Transferrin receptor 1 (TfR1) is highly expressed in brain capillary endothelial cells and TMZ-resistant glioma cells. In this study, OXA was loaded into ferritin (Fn) to prepare glioma-targeted oxaliplatin/ferritin clathrate OXA@Fn. OXA@Fn efficiently crossed BBB and was actively taken up by TMZ-resistant glioma cells via TfR1. Then, OXA increased the intracellular H2O2 level and induced the apoptosis of TMZ-resistant glioma cells. Meanwhile, Fn increased Fe2+ level in TMZ-resistant glioma cells. In addition, the expression of ferroportin 1 was significantly reduced, resulting in Fe2+ to be locked up inside the TMZ-resistant glioma cells. This subsequently enhanced the Fenton reaction and boosted the ferroptosis of TMZ-resistant glioma cells. Consequently, T cell mediated anti-tumor immune response was strongly induced, and the immunosuppressive microenvironment was significantly reversed in TMZ-resistant glioma tissue. Ultimately, the growth and invasion of TMZ-resistant glioma was inhibited by OXA@Fn. OXA@Fn shows great potential in the treatment of TMZ-resistant glioma and prospect in clinical transformation.Graphical

Involvement of Astrocytes in the Formation, Maintenance, and Function of the Blood-Brain Barrier.

The blood-brain barrier (BBB) is a fundamental structure that protects the composition of the brain by determining which ions, metabolites, and nutrients are allowed to enter the brain from the blood or to leave it towards the circulation. The BBB is structurally composed of a layer of brain capillary endothelial cells (BCECs) bound to each other through tight junctions (TJs). However, its development as well as maintenance and properties are controlled by the other brain cells that contact the BCECs: pericytes, glial cells, and even neurons themselves. Astrocytes seem, in particular, to have a very important role in determining and controlling most properties of the BBB. Here, we will focus on these latter cells, since the comprehension of their roles in brain physiology has been continuously expanding, even including the ability to participate in neurotransmission and in complex functions such as learning and memory. Accordingly, pathological conditions that alter astrocytic functions can alter the BBB's integrity, thus compromising many brain activities. In this review, we will also refer to different kinds of in vitro BBB models used to study the BBB's properties, evidencing its modifications under pathological conditions.

Physiological and Pathological Significance of Chloride Channels.

Cl- influx and efflux through Cl- channels play a role in regulating the homeostasis of biological functions. Therefore, the hyperfunction or dysfunction of Cl- channels elicits pathological mechanisms. The Cl- channel superfamily includes voltage-gated Cl- (ClC) channels, Ca2+-activated Cl- channels (ClCa; TMEM16A/TMEM16B), cystic fibrosis transmembrane conductance regulator channels, and ligand-gated Cl- channels. These channels are ubiquitously expressed to regulate ion homeostasis, muscle tonus, membrane excitability, cell volume, survival, neurotransmission, and transepithelial transport. The activation or inhibition of Cl- channels changes the membrane potential, thereby affecting cytosolic Ca2+ signals. An elevation in cytosolic [Ca2+] triggers physiological and pathological responses in most cells. However, the roles of Cl- channels have not yet been examined as extensively as cation (Na+, Ca2+, and K+) channels. We recently reported the functional expression of: (i) TMEM16A/ClCa channels in portal vein and pulmonary arterial smooth muscle cells (PASMC), pinealocytes, and brain capillary endothelial cells; (ii) TMEM16B/ClCa channels in pinealocytes; (iii) ClC-3 channels in PASMC and chondrocytes; and (iv) ClC-7 channels in chondrocytes. We also showed that the down-regulation of TMEM16A and ClC-7 channel expression was associated with cirrhotic portal hypertension and osteoarthritis, respectively, whereas the enhanced expression of TMEM16A and ClC-3 channels was involved in the pathogenesis of cerebral ischemia and pulmonary arterial hypertension, respectively. Further investigations on the physiological/pathological functions of Cl- channels will provide insights into biological functions and contribute to the screening of novel target(s) of drug discovery for associated diseases.

Virus-Inspired Glucose and Polydopamine (GPDA)-Coating as an Effective Strategy for the Construction of Brain Delivery Platforms.

The ability of drugs to cross the blood-brain barrier (BBB) is crucial for treating central nervous system (CNS) disorders. Inspired by natural viruses, here we report a glucose and polydopamine (GPDA) coating method for the construction of delivery platforms for efficient BBB crossing. Such platforms are composed of nanoparticles (NPs) as the inner core and surface functionalized with glucose-poly(ethylene glycol) (Glu-PEG) and polydopamine (PDA) coating. Glu-PEG provides selective targeting of the NPs to brain capillary endothelial cells (BCECs), while PDA enhances the transcytosis of the NPs. This strategy is applicable to gold NPs (AuNPs), silica, and polymeric NPs, which achieves as high as 1.87% of the injected dose/g of brain in healthy brain tissues. In addition, the GPDA coating manages to deliver NPs into the tumor tissue in the orthotopic glioblastoma model. Our study may provide a universal strategy for the construction of delivery platforms for efficient BBB crossing and brain drug delivery.

MMP-2-mediated Scube2 degradation promotes blood-brain barrier disruption by blocking the interaction between astrocytes and endothelial cells via inhibiting Sonic hedgehog pathway during early cerebral ischemia.

We have previously demonstrated a rapid secretion of matrix metalloproteinase-2 (MMP-2) in the ischemic brain. Since Scube2 can interact with Sonic hedgehog (Shh) to maintain blood-brain barrier (BBB) integrity via regulating the interaction between brain capillary endothelial cells (ECs) and perivascular astrocytes, and it is also a substrate of MMP-2, we hypothesized that the secreted MMP-2 could degrade Scube2 and contribute to ischemic BBB disruption. Using an invitro ischemic model of 90-min oxygen-glucose deprivation/3-h reoxygenation (OGD/R) and an invivo mouse stroke model of 90-min middle cerebral artery occlusion (MCAO) with 3-h reperfusion, we established an important role of MMP-2-mediated Scube2 degradation in early ischemic BBB disruption. Exposure of C8-D1A cells and bEnd.3 cells to OGD/R increased MMP secretion in both cells, and C8-D1A cells appeared to secrete more MMPs than bEnd.3 cells. Co-IP and double-immunostaining revealed that Scube2 co-localized well with MMP-2 in C8-D1A cells and could be pulled down by MMP-2 antibodies. In MCAO mice, Scube2 protein showed a drastic reduction in ischemic brain tissue, which was accompanied by suppressed expression of Shh and its downstream molecules. Of note, specific knockdown of astrocytic Scube2 with AAV-shScube2 augmented MCAO-induced Shh suppression and exacerbated BBB leakage and inflammatory reactions in the ischemic brain. Last, incubation of bEnd.3 cells with conditioned medium derived from OGD-treated C8-D1A cells led to a significant inhibition of the Shh pathway in bEnd.3 cells and degradation of VE-cadherin and ZO-1. Inhibition of MMP-2 with SB-3CT or over-expression of Scube2 with plasmids in C8-D1A cells alleviated the above effect of C8-D1A cells-derived conditioned medium. Taken together, our data indicate that ischemia-induced secretion of MMP-2 may contribute to early BBB disruption in ischemic stroke via interrupting the shared Scube2-Shh pathway between brain capillary ECs and perivascular astrocytes.

Prednisolone Targets Claudins in Mouse Brain Blood Vessels.

Endothelial cells in brain capillaries are crucial for the function of the blood-brain barrier (BBB), and members of the tight junction protein family of claudins are regarded to be primarily responsible for barrier properties. Thus, the analysis of bioactive substances that can affect the BBB's permeability is of great importance and may be useful for the development of new therapeutic strategies for brain pathologies. In our study, we tested the hypothesis that the application of the glucocorticoid prednisolone affects the murine blood-brain barrier in vivo. Isolated brain tissue of control and prednisolone-injected mice was examined by employing immunoblotting and confocal laser scanning immunofluorescence microscopy, and the physiological and behavioral effects were analyzed. The control tissue samples revealed the expression of barrier-forming tight junction proteins claudin-1, -3, and -5 and of the paracellular cation and water-channel-forming protein claudin-2. Prednisolone administration for 7 days at doses of 70 mg/kg caused physiological and behavioral effects and downregulated claudin-1 and -3 and the channel-forming claudin-2 without altering their localization in cerebral blood vessels. Changes in the expression of these claudins might have effects on the ionic and acid-base balance in brain tissue, suggesting the relevance of our findings for therapeutic options in disorders such as cerebral edema and psychiatric failure.

Characterization of an iPSC-based barrier model for blood-brain barrier investigations using the SBAD0201 stem cell line

BackgroundBlood-brain barrier (BBB) models based on primary murine, bovine, and porcine brain capillary endothelial cell cultures have long been regarded as robust models with appropriate properties to examine the functional transport of small molecules. However, species differences sometimes complicate translating results from these models to human settings. During the last decade, brain capillary endothelial-like cells (BCECs) have been generated from stem cell sources to model the human BBB in vitro. The aim of the present study was to establish and characterize a human BBB model using human induced pluripotent stem cell (hiPSC)-derived BCECs from the hIPSC line SBAD0201.MethodsThe model was evaluated using transcriptomics, proteomics, immunocytochemistry, transendothelial electrical resistance (TEER) measurements, and, finally, transport assays to assess the functionality of selected transporters and receptor (GLUT-1, LAT-1, P-gp and LRP-1).ResultsThe resulting BBB model displayed an average TEER of 5474 ± 167 Ω·cm2 and cell monolayer formation with claudin-5, ZO-1, and occludin expression in the tight junction zones. The cell monolayers expressed the typical BBB markers VE-cadherin, VWF, and PECAM-1. Transcriptomics and quantitative targeted absolute proteomics analyses revealed that solute carrier (SLC) transporters were found in high abundance, while the expression of efflux transporters was relatively low. Transport assays using GLUT-1, LAT-1, and LRP-1 substrates and inhibitors confirmed the functional activities of these transporters and receptors in the model. A transport assay suggested that P-gp was not functionally expressed in the model, albeit antibody staining revealed that P-gp was localized at the luminal membrane.ConclusionsIn conclusion, the novel SBAD0201-derived BBB model formed tight monolayers and was proven useful for studies investigating GLUT-1, LAT-1, and LRP-1 mediated transport across the BBB. However, the model did not express functional P-gp and thus is not suitable for the performance of drug efflux P-gp reletated studies.

Anchoring Microbubbles on Cerebrovascular Endothelium as a New Strategy Enabling Low-Energy Ultrasound-Assisted Delivery of Varisized Agents Across Blood-Brain Barrier.

The protective blood-brain barrier (BBB) prevents most therapeutic agents from entering the brain. Currently, focused ultrasound (FUS) is mostly employed to create microbubbles that induce a cavitation effect to open the BBB. However, microbubbles pass quickly through brain microvessels, substantially limiting the cavitation effect. Here, we constructed a novel perfluoropropane-loaded microbubble, termed ApoER-Pep-MB, which possessed a siloxane bonds-crosslinked surface to increase the microbubble stability against turbulence in blood circulation and was decorated with binding peptide for apolipoprotein E receptor (ApoER-Pep). The microbubble with tailor-made micron size (2µm) and negative surface charge (-30mV) performed ApoER-mediated binding rather than internalization into brain capillary endothelial cells. Consequently, the microbubble accumulated on the brain microvessels, based on which even a low-energy ultrasound with less safety risk than FUS, herein diagnostic ultrasound (DUS), could create a strong cavitation effect to open the BBB. Evans Blue and immunofluorescence staining studies demonstrated that the DUS-triggered cavitation effect not only temporarily opened the BBB for 2 h but also caused negligible damage to the brain tissue. Therefore, various agents, ranging from small molecules to nanoscale objects, can be efficiently delivered to target regions of the brain, offering tremendous opportunities for the treatment of brain diseases.