Brain Endothelial Cells Research Articles

Recent advances in drug delivery and targeting to the brain.

Our body keeps separating the toxic chemicals in the blood from the brain. A significant number of drugs do not enter the central nervous system (CNS) due to the blood-brain barrier (BBB). Certain diseases, such as tumor growth and stroke, are known to increase the permeability of the BBB. However, the heterogeneity of this permeation makes it difficult and unpredictable to transport drugs to the brain. In recent years, research has been directed toward increasing drug penetration inside the brain, and nanomedicine has emerged as a promising approach. Active targeting requires one or more specific ligands on the surface of nanoparticles (NPs), which brain endothelial cells (ECs) recognize, allowing controlled drug delivery compared to conventional targeting strategies. This review highlights the mechanistic insights about different cell types contributing to the development and maintenance of the BBB and summarizes the recent advancement in brain-specific NPs for different pathological conditions. Furthermore, fundamental properties of brain-targeted NPs will be discussed, and the standard lesion features classified by neurological pathology are summarized.

INCREASED EICOSAPENTAENOIC ACID TO ARACHIDONIC ACID (EPA/AA) RATIO IN BRAIN ENDOTHELIAL CELLS ASSOCIATED WITH REDUCED EXPRESSION OF NEUTROPHIL DEGRANULATION PROTEINS DURING INFLAMMATION

Endothelial cell (EC) dysfunction results in inflammation and increased atherothrombotic risk, including stroke. The omega-3 fatty acid (FA) eicosapentaenoic acid (EPA) improves arterial compliance in a manner predicted by circulating EPA/AA ratios, an indicator of cardiovascular (CV) risk. Treatment with icosapent ethyl (IPE), a formulation of highly purified EPA, reduced composite CV events, including stroke, in REDUCE-IT that correlated with on-treatment EPA levels. We measured the effects of EPA on the EPA/AA ratio and protein expression in endothelial cells from brain ECs (BECs) following cytokine challenge.

Role of NADPH Oxidases in Blood-Brain Barrier Disruption and Ischemic Stroke.

NADPH oxidases (Nox) are one of the main sources of reactive oxygen species (ROS) in the central nervous system (CNS). While these enzymes have been shown to be involved in physiological regulation of cerebral vascular tone, excessive ROS produced by Nox1-5 play a critical role in blood–brain barrier (BBB) dysfunction in numerous neuropathologies. Nox-derived ROS have been implicated in mediating matrix metalloprotease (MMP) activation, downregulation of junctional complexes between adjacent brain endothelial cells and brain endothelial cell apoptosis, leading to brain microvascular endothelial barrier dysfunction and consequently, increases in BBB permeability. In this review, we will highlight recent findings on the role played by these enzymes in BBB disruption induced by ischemic stroke.

High-Throughput Screening Assay Identifies Berberine and Mubritinib as Neuroprotection Drugs for Spinal Cord Injury via Blood-Spinal Cord Barrier Protection.

Because the breakdown of the blood-brain spinal cord barrier (BBSCB) worsens many central nervous system (CNS) diseases, prevention of BBSCB breakdown has been a major therapeutic target, especially for spinal cord injury (SCI). However, effective drugs that protect BBSCB function have yet to be developed. The purpose of the current study was 1) to develop a high-throughput screening assay (HTSA) to identify candidate drugs to protect BBSCB function, 2) to identify candidate drugs from existing drugs with newly developed HTSA, and 3) to examine the therapeutic effects of candidate drugs on SCI. Our HTSA included a culture of immortalized human brain endothelial cells primed with candidate drugs, stress with H2O2, and evaluation of their viability. A combination of the resazurin-based assay with 0.45mM H2O2 qualified as a reliable HTSA. Screening of 1,570 existing drugs identified 90 drugs as hit drugs. Through a combination of reproducibility tests, exclusion of drugs inappropriate for clinical translation, and dose dependency tests, berberine, mubritinib, and pioglitazone were identified as a candidate. An in vitro BBSCB functional test revealed that berberine and mubritinib, but not pioglitazone, protected BBSCB from oxygen-glucose deprivation and reoxygenation stress. Additionally, these two drugs minimized BBSCB breakdown 1day after cervical SCI in mice. Furthermore, berberine and mubritinib reduced neuronal loss and improved gait performance 8weeks after SCI. Collectively, the current study established a useful HTSA to identify potential neuroprotective drugs by maintaining BBSCB function and demonstrated the neuroprotective effect of berberine and mubritinib after SCI.

Exosomes derived from M2 Macrophages Improve Angiogenesis and Functional Recovery after Spinal Cord Injury through HIF-1α/VEGF Axis.

Exosomes are nano-sized vesicles that contain a variety of mRNAs, miRNAs, and proteins. They are capable of being released by a variety of cells and are essential for cell–cell communication. The exosomes produced by cells have shown protective benefits against spinal cord damage (SCI). Recently, it was discovered that M2 macrophages aid in the angiogenesis of numerous illnesses. However, the functional role of M2 macrophage-derived exosomes on SCI is unclear. Here, we investigate the pro-angiogenesis of M2 macrophage-derived exosomes on SCI. We founded that M2 macrophage exosomes alleviated tissue damage and enhanced functional recovery post-SCI. We discovered that M2 macrophage exosome administration increased angiogenesis after SCI in vivo using immunohistochemistry, immunofluorescence labeling, and Western blot analysis. Additionally, the expression of the pro-angiogenesis factors, HIF-1α and VEGF, were enhanced with the treatment of the M2 macrophage exosomes. Furthermore, we found that M2 macrophage exosomes enhanced neurogenesis after SCI in vivo. In vitro, we found that M2 macrophage exosomes can be taken up by the brain endothelial cell line (bEnd.3) and that they enhanced the tube formation, migration, and proliferation of bEnd.3 cells. Furthermore, by using special siRNA to inhibit HIF-1α expression, we observed that the expression of VEGF decreased, and the tube formation, migration, and proliferation of bEnd.3 cells were attenuated with the treatment of HIF-1α-siRNA. In conclusion, our findings reveal that M2 macrophage exosomes improve neurological functional recovery and angiogenesis post-SCI, and this process is partially associated with the activation of the HIF-1/VEGF signaling pathway.

Brain Endothelial Cells in Contrary to the Aortic Do Not Transport but Degrade Low-Density Lipoproteins via Both LDLR and ALK1.

The transport of low-density lipoprotein (LDL) through the endothelium is a key step in the development of atherosclerosis, but it is notorious that phenotypic differences exist between endothelial cells originating from different vascular beds. Endothelial cells forming the blood–brain barrier restrict paracellular and transcellular passage of plasma proteins. Here, we systematically compared brain versus aortic endothelial cells towards their interaction with LDL and the role of proteins known to regulate the uptake of LDL by endothelial cells. Both brain endothelial cells and aortic endothelial cells bind and internalize LDL. However, whereas aortic endothelial cells degrade very small amounts of LDL and transcytose the majority, brain endothelial cells degrade but do not transport LDL. Using RNA interference (siRNA), we found that the LDLR–clathrin pathway leads to LDL degradation in either endothelial cell type. Both loss- and gain-of-function experiments showed that ALK1, which promotes transcellular LDL transport in aortic endothelial cells, also limits LDL degradation in brain endothelial cells. SR-BI and caveolin-1, which promote LDL uptake and transport into aortic endothelial cells, limit neither binding nor association of LDL to brain endothelial cells. Together, these results indicate distinct LDL trafficking by brain microvascular endothelial cells and aortic endothelial cells.

Optimized protocol for translatome analysis of mouse brain endothelial cells.

Brain endothelial cells (BECs) are important conduits that deliver oxygen and nutrients, protect parenchyma cells from toxins, and drain wastes to maintain brain homeostasis. Impairment of BECs has been implicated in diverse neurodegenerative diseases, including Alzheimer’s disease and Parkinson’s disease. Therefore, molecular analysis of BECs is important for understanding the molecular pathogenesis of these neurological diseases. Even though many transcriptome analyses for BECs have been developed, mRNA levels do not necessarily correlate with the levels of actively translated proteins. Translatome analysis using RiboTag mice, in which Rpl22, a ribosomal component, is tagged by the hemagglutinin epitope under Cre recombinase activation, could serve as an excellent tool that overcomes these caveats. However, implementation of this technique is limited by high noise-to-signal ratios as well as the low yield of mRNAs from BECs, which limits bulk gene expression analysis. In this study, we established a protocol to isolate highly pure mRNAs from BECs in the cortex of eight- to twelve-week-old male Tie2-Cre; Rpl22HA/HA mice by using a cell strainer to trap blood vessels prior to immunoprecipitation. According to the results of RT–PCR, the specificity of the mRNA pools isolated by our protocol was much higher than that of the pools isolated by the standard protocol. We were also able to generate a high-quality cDNA library for RNA-seq with the small amount of mRNA isolated with our protocol. Thus, this optimized method will be useful for future studies of BECs at the molecular level.

SZN-413, a FZD4 Agonist, as a Potential Novel Therapeutic for the Treatment of Diabetic Retinopathy.

PurposeThere remains a high unmet need for therapies with new mechanisms of action to achieve reperfusion of ischemic retina in diabetic retinopathy. We examined whether a novel frizzled class receptor 4 (FZD4) agonist could promote regeneration of functional blood vessels in animal models of retinopathy.MethodsWe developed a novel Norrin mimetic (SZN-413-p) targeting FZD4 and low-density lipoprotein receptor-related protein 5 (LRP5) and examined its effect on retinal and brain endothelial cells in vitro. SZN-413-p was subsequently humanized, resulting in the therapeutic candidate SZN-413, and was examined in animal models of retinopathy. In an oxygen-induced retinopathy mouse model, avascular and neovascularization areas were measured. Furthermore, in a vascular endothelial growth factor (VEGF)-induced retinal vascular leakage rabbit model, the impact on vascular leakage by SZN-413 was examined by measuring fluorescein leakage.ResultsSZN-413-p induced Wnt/β-catenin signaling and upregulated blood–brain barrier/blood–retina barrier gene expressions in endothelial cells. In the oxygen-induced retinopathy mouse model, SZN-413-p and SZN-413 significantly reduced the neovascularization area size (P < 0.001) to a level comparable to, or better than the positive control aflibercept. Both agonists also showed a reduction in avascular area size compared to vehicle (P < 0.001) and aflibercept groups (P < 0.05 and P < 0.01 for SZN-413-p and SZN-413, respectively). In the VEGF-induced retinal vascular leakage rabbit model, SZN-413 reduced retinal vascular leakage by ∼80%, compared to the vehicle-treated group (P < 0.01).ConclusionsReduction of neovascular tufts and avascular areas and of VEGF-driven retinal vascular leakage suggests that SZN-413 can simultaneously address retinal non-perfusion and vascular leakage.Translational RelevanceFZD4 signaling modulation by SZN-413 is a novel mechanism of action that can offer a new therapeutic strategy for diabetic retinopathy.

Exosomes form tunneling nanotubes (TUNTs) in the blood-brain barrier: a nano-anatomical perspective of barrier genesis.

The blood-brain barrier (BBB) is a robust interface between the blood and the central nervous system. Barrier type endothelium is able to limit paracellular (PC) movement, relegating molecular flux to the transendothelial pathways of brain endothelial cells (BECs). It is, therefore, apparent that any leakage via the PC shunts would effectively nullify the regulation of molecular flux across the transcellular pathways. The application of higher-resolution scanning electron microscopy (HR-SEM) illuminates the heterogenous, morphological profile that exists on the surface of BEC membranes and the relationship between these ultrastructures during the molecular construction of the PC space between adjacent BECs. In this study developing BEC monolayers were grown on mixed, cellulose esters insert membranes in a bicameral system. BEC monolayers were fixed in 2.5% glutaraldehyde, hydrated, critically dried, and sputter-coated, for imaging utilizing HR-SEM. This study, for the first time, showed membrane-bound exosomes were attached to the plasma membrane surfaces of the BECs. The exosomes were characterized as small membrane-bound, nano-sized exosomes (30–300 nm). Based on their membrane morphology and anatomical structure, exosomes appear to possess two distinct functions, namely: paracrine secretion and nanotube construction between adjacent BECs, during in vitro barrier genesis. The HR-SEM micrographs in conjunction with the Tipifarnib inhibition of exosome formation, suggests that brain capillary endothelial exosomes play a prominent role in the bilateral signaling, which contribute to the regulation of the permeability of the BBB. Given that blood-brain barrier permeability has been implicated in the progression of many neurodegenerative pathologies, the role of these exosomes and TUNTs posits the capacity of these structures to exacerbate neuropathologies that implicate BBB permeability. These findings could lead to the development of novel treatment interventions and moreover, the characterization of BBB exosomes may be a reliable target for identifying therapeutic biomarkers in neurodegenerative disease. Conversely, the presence of BBB exosomes raises a critical enterprise to target the exosome-induced nanotubes as a vehicle for transferring therapeutic treatments across the BBB.

The impaired distribution of adenosine deaminase isoenzymes in multiple sclerosis plasma and cerebrospinal fluid.

BackgroundAdenosine deaminase (ADA) via two isoenzymes, ADA1 and ADA2, regulates intra- and extracellular adenosine concentrations by converting it to inosine. In the central nervous system (CNS), adenosine modulates the processes of neuroinflammation and demyelination that together play a critical role in the pathophysiology of multiple sclerosis (MS). Except for their catalytic activities, ADA isoenzymes display extra-enzymatic properties acting as an adhesion molecule or a growth factor.AimsThis study aimed to explore the distribution and activity of ADA1 and ADA2 in the plasma and the CSF of MS patients as well as in the human brain microvascular endothelial cells (HBMEC), human brain vascular pericytes and human astrocytes.Methods and resultsThe enzyme assay following reverse phase-high performance liquid chromatography (HPLC) analysis was used to detect the ADA1 and ADA2 activities and revealed an increased ratio of ADA1 to ADA2 in both the plasma and the CSF of MS patients. Plasma ADA1 activity was significantly induced in MS, while ADA2 was decreased in the CSF, but significance was not reached. The brain astrocytes, pericytes and endothelial cells revealed on their surface the activity of ADA1, with its basal level being five times higher in the endothelial cells than in the astrocytes or the pericytes. In turn, ADA2 activity was only observed in pericytes and endothelial cells. Stimulation of the cells with pro-inflammatory cytokines TNFα/IL17 for 18 h decreased intracellular nucleotide levels measured by HPLC only in pericytes. The treatment with TNFα/IL17 did not modulate cell-surface ATP and AMP hydrolysis nor adenosine deamination in pericytes or astrocytes. Whereas in endothelial cells it downregulated AMP hydrolysis and ADA2 activity and upregulated the ADA1, which reflects the ADA isoenzyme pattern observed here in the CSF of MS patients.ConclusionIn this study, we determined the impaired distribution of both ADA isoenzymes in the plasma and the CSF of patients with MS. The increased ADA1 to ADA2 ratio in the CSF and plasma may translate to unfavorable phenotype that triggers ADA1-mediated pro-inflammatory mechanisms and decreases ADA2-dependent neuroprotective and growth-promoting effects in MS.

Neuropilin-1 modulates the 3D invasive properties of glioblastoma stem-like cells

Glioblastoma multiforme (GBM) is a rare, yet devastating, primary brain tumor in adults. Current treatments remain generally ineffective and GBM almost invariably recurs, resulting in median survival of 15 months. This high malignancy sources notably from the resilience and invasive capabilities of tumor cells. Within GBM, exists a population of self-sustaining transformed cells with stem-like properties (GSCs), which are thought to be responsible for tumor initiation, growth, and invasion, as well as recurrence. In the tumor microenvironment, GSCs might be found in the vicinity of brain endothelial cells, which provide a protective habitat. Likewise, these resistant, quiescent GSCs may accumulate in hypoxic zones, away from the perivascular niche, or travel towards the healthy brain parenchyma, by eminently co-opting neuro-vascular tracks. Herein, we established an ex vivo model to explore GSC invasive behavior. We found that patient-derived cells massively invade the collagen matrix. In addition, we described that the glycoprotein Neuropilin-1 (NRP1) contributes to GSC spreading and invasion. Indeed, both RNA interference-mediated silencing and CRISPR-mediated gene editing deletion of NRP1 strongly impaired the 3D invasive properties of patient-derived GSCs and their close localization to the brain blood vessels. Of note, other typical features of GSCs, such as expansion and self-renewal were maintained. From a mechanistic standpoint, this biological effect might rely on the expression of the β3 subunit integrin cell-extracellular matrix adhesive receptor. Our data, therefore, propose a reliable approach to explore invasive properties of patient glioma cells ex vivo and identify NRP1 as a mediator in this malignant process.

Engineering Extracellular Vesicles to Modulate Their Innate Mitochondrial Load.

Extracellular vesicles (EVs) are promising carriers for the delivery of biotherapeutic cargo such as RNA and proteins. We have previously demonstrated that the innate EV mitochondria in microvesicles (MVs), but not exosomes (EXOs) can be transferred to recipient BECs and mouse brain slice neurons. Here, we sought to determine if the innate EV mitochondrial load can be further increased via increasing mitochondrial biogenesis in the donor cells. We hypothesized that mitochondria-enriched EVs ("mito-EVs") may increase the recipient BEC ATP levels to a greater extent than naïve MVs. We treated NIH/3T3, a fibroblast cell line and hCMEC/D3, a human brain endothelial cell (BEC) line using resveratrol to activate peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC-1α), the central mediator of mitochondrial biogenesis. Naïve EVs and mito-EVs isolated from the non-activated and activated donor cells were characterized using transmission electron microscopy, dynamic light scattering and nanoparticle tracking analysis. The effect of mito-EVs on resulting ATP levels in the recipient BECs were determined using Cell Titer Glo ATP assay. The uptake of Mitotracker Red-stained EVs into recipient BECs and their colocalization with recipient BEC mitochondria were studied using flow cytometry and fluorescence microscopy. Resveratrol treatment increased PGC-1α expression in the donor cells. Mito-MVs but not mito-EXOs showed increased expression of mitochondrial markers ATP5A and TOMM20 compared to naïve MVs. TEM images showed that a greater number of mito-MVs contained mitochondria compared to naïve MVs. Mito-MVs but not mito-EXOs showed a larger particle diameter compared to their naïve EV counterparts from the non-activated cells suggesting increased mitochondria incorporation. Mito-EVs were generated at higher particle concentrations compared to naïve EVs from non-activated cells. Mito-EVs increased the cellular ATP levels and transferred their mitochondrial load into the recipient BECs. Mito-MV mitochondria also colocalized with recipient BEC mitochondria. Our results suggest that the pharmacological modulation of mitochondrial biogenesis in the donor cells can change the mitochondrial load in the secreted MVs. Outcomes of physicochemical characterization studies and biological assays confirmed the superior effects of mito-MVs compared to naïve MVs-suggesting their potential to improve mitochondrial function in neurovascular and neurodegenerative diseases. The online version contains supplementary material available at 10.1007/s12195-022-00738-8.

Substance P Reduces Infarct Size and Mortality After Ischemic Stroke, Possibly Through the M2 Polarization of Microglia/Macrophages and Neuroprotection in the Ischemic Rat Brain.

Substance-P (SP) is an 11 amino acid neuropeptide that is known to stimulate the peripheral mobilization of bone marrow mesenchymal stem cells and M2 polarization in monocytes/macrophages in a variety of acute and chronic tissue injuries. To examine the role of SP in protection and recovery from acute ischemic brain injury, experimental ischemic stroke was induced by transient middle cerebral artery occlusion (tMCAo) in rats for 1h with subsequent reperfusion. Two injections of SP, immediately and one day post-tMCAo, resulted in approximately threefold lower mortality and 40% less infarct volume than those of saline-treated rats at seven days post-tMCAo. At 4.5h, SP markedly increased CD11b/c+CD163+/CD 206+ cells in the blood, which were concomitantly decreased in the bone marrow, suggesting that SP preferentially mobilized M2-polarized monocytes. After two days, SP increased the expression of neuroprotective and anti-inflammatory genes in the ischemic brain and induced neuronal survival in the brain penumbra. Additionally, SP markedly increased CD68+CD163+ and CD68+CD206+ M2 microglia/macrophages in the ischemic brain during seven days post-tMCAo. Furthermore, SP preserved the blood‒brain barrier in the ischemic brain, which was confirmed by the abundant levels of SMI71+ brain endothelial cells that colocalized with α-SMA+ pericytes. The beneficial effects of SP on functional recovery and tissue preservation were maintained for six weeks. Collectively, SP treatment in the early phase of ischemic stroke markedly suppressed the destructive inflammatory response and improved the microenvironment for tissue protection and repair.

CLMP Promotes Leukocyte Migration Across Brain Barriers in Multiple Sclerosis.

Background and ObjectivesIn multiple sclerosis (MS), peripheral immune cells use various cell trafficking molecules to infiltrate the CNS where they cause damage.The objective of this study was to investigate the involvement of coxsackie and adenovirus receptor–like membrane protein (CLMP) in the migration of immune cells into the CNS of patients with MS.MethodsExpression of CLMP was measured in primary cultures of human brain endothelial cells (HBECs) and human meningeal endothelial cells (HMECs), postmortem brain samples, and peripheral blood mononuclear cells (PBMCs) from patients with MS and controls by RNA sequencing, quantitative PCR, immunohistochemistry, and flow cytometry. In vitro migration assays using HBECs and HMECs were performed to evaluate the function of CLMP.ResultsUsing bulk RNA sequencing of primary cultures of human brain and meningeal endothelial cells (ECs), we have identified CLMP as a new potential cell trafficking molecule upregulated in inflammatory conditions. We first confirmed the upregulation of CLMP at the protein level on TNFα-activated and IFNγ-activated primary cultures of human brain and meningeal ECs. In autopsy brain specimens from patients with MS, we demonstrated an overexpression of endothelial CLMP in active MS lesions when compared with normal control brain tissue. Flow cytometry of human PBMCs demonstrated an increased frequency of CLMP+ B lymphocytes and monocytes in patients with MS, when compared with that in healthy controls. The use of a blocking antibody against CLMP reduced the migration of immune cells across the human brain and meningeal ECs in vitro. Finally, we found CLMP+ immune cell infiltrates in the perivascular area of parenchymal lesions and in the meninges of patients with MS.DiscussionCollectively, our data demonstrate that CLMP is an adhesion molecule used by immune cells to access the CNS during neuroinflammatory disorders such as MS. CLMP could represent a target for a new treatment of neuroinflammatory conditions.

TNF-α and IL-1β Modulate Blood-Brain Barrier Permeability and Decrease Amyloid-β Peptide Efflux in a Human Blood-Brain Barrier Model.

The blood-brain barrier (BBB) is a selective barrier and a functional gatekeeper for the central nervous system (CNS), essential for maintaining brain homeostasis. The BBB is composed of specialized brain endothelial cells (BECs) lining the brain capillaries. The tight junctions formed by BECs regulate paracellular transport, whereas transcellular transport is regulated by specialized transporters, pumps and receptors. Cytokine-induced neuroinflammation, such as the tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), appear to play a role in BBB dysfunction and contribute to the progression of Alzheimer’s disease (AD) by contributing to amyloid-β (Aβ) peptide accumulation. Here, we investigated whether TNF-α and IL-1β modulate the permeability of the BBB and alter Aβ peptide transport across BECs. We used a human BBB in vitro model based on the use of brain-like endothelial cells (BLECs) obtained from endothelial cells derived from CD34+ stem cells cocultivated with brain pericytes. We demonstrated that TNF-α and IL-1β differentially induced changes in BLECs’ permeability by inducing alterations in the organization of junctional complexes as well as in transcelluar trafficking. Further, TNF-α and IL-1β act directly on BLECs by decreasing LRP1 and BCRP protein expression as well as the specific efflux of Aβ peptide. These results provide mechanisms by which CNS inflammation might modulate BBB permeability and promote Aβ peptide accumulation. A future therapeutic intervention targeting vascular inflammation at the BBB may have the therapeutic potential to slow down the progression of AD.

Activation of NMDA receptors in brain endothelial cells increases transcellular permeability

Neurovascular coupling is a precise mechanism that induces increased blood flow to activated brain regions, thereby providing oxygen and glucose. In this study, we hypothesized that N-methyl-D-aspartate (NMDA) receptor signaling, the most well characterized neurotransmitter signaling system which regulates delivery of essential molecules through the blood–brain barrier (BBB). Upon application of NMDA in both in vitro and in vivo models, increased delivery of bioactive molecules that was mediated through modulation of molecules involved in molecular delivery, including clathrin and caveolin were observed. Also, NMDA activation induced structural changes in the BBB and increased transcellular permeability that showed regional heterogeneity in its responses. Moreover, NMDA receptor activation increased endosomal trafficking and facilitated inactivation of lysosomal pathways and consequently increased molecular delivery mediated by activation of calmodulin-dependent protein kinase II (CaMKII) and RhoA/protein kinase C (PKC). Subsequent in vivo experiments using mice specifically lacking NMDA receptor subunit 1 in endothelial cells showed decreased neuronal density in the brain cortex, suggesting that a deficiency in NMDA receptor signaling in brain endothelial cells induces neuronal losses. Together, these results highlight the importance of NMDA-receptor-mediated signaling in the regulation of BBB permeability that surprisingly also affected CD31 staining.

Pro-angiogenic activity of isofuran.

Isofurans (IsoFs) are a series of novel discovered lipid peroxidation products. This study focused on the investigation of the angiogenic property of IsoF. MTT stain assay indicated that 1 µm IsoF had the most bioactivity in rat brain endothelial cells (RBECs). IsoF significantly promoted cellular proliferation and migration and remarkably decreased staurosporine-induced apoptosis by TUNEL assay in the RBECs. It successfully up-regulated rat aortic vascularization and choroid explant sprouting, extracellular regulated protein kinases (ERK)1/2, and triggered calcium release. RT-PCR examination indicated that IsoF up-regulated tumor necrosis factor (TNF)α, angiopoietin-1 receptor (Tie2), and vascular endothelial growth factor (VEGF)-A, but did not interfere with caspase 2 and VEGF-C in the RBECs. IsoF has pro-angiogenic activity. Calcium release and ERK1/2 phosphorylation may be involved in the signaling of the IsoF-induced up-regulation of TNFα, Tie2, and VEGF-A, which could be the molecular mechanism of the pro-angiogenic activity of the IsoF.

Altered blood-brain barrier transport of nanotherapeutics in lysosomal storage diseases.

Treatment of neurological lysosomal storage disorders (LSDs) are limited because of impermeability of the blood-brain barrier (BBB) to macromolecules. Nanoformulations targeting BBB transcytosis are being explored, but the status of these routes in LSDs is unknown. We studied nanocarriers (NCs) targeted to the transferrin receptor (TfR), ganglioside GM1 or ICAM1, associated to the clathrin, caveolar or cell adhesion molecule (CAM) routes, respectively. We used brain endothelial cells and mouse models of acid sphingomyelinase-deficient Niemann Pick disease (NPD), and postmortem LSD patients' brains, all compared to respective controls. NC transcytosis across brain endothelial cells and brain distribution in mice were affected, yet through different mechanisms. Reduced TfR and clathrin expression were found, along with decreased transcytosis in cells and mouse brain distribution. Caveolin-1 expression and GM1 transcytosis were also reduced, yet increased GM1 levels seemed to compensate, providing similar NC brain distribution in NPD vs. control mice. A tendency to lower NHE-1 levels was seen, but highly increased ICAM1 expression in cells and human brains correlated with increased transcytosis and brain distribution in mice. Thus, transcytosis-related alterations in NPD and likely other LSDs may impact therapeutic access to the brain, illustrating the need for these mechanistic studies.

The role of glutathione conjugation on the transcellular transport process of PEGylated liposomes across the blood brain barrier

Notwithstanding the growing evidence of improved drug delivery efficiency to the brain by ligand modification of PEGylated liposomes, the comprehensive knowledge of their transport processes and payload across the BBB is yet to be revealed. Herein, this study sought to understand the glutathione (GSH) ligand effect on transcellular transport mechanisms of liposomes through the blood–brain barrier (BBB) by comparing PEGylated liposomes (PEG-L) and GSH PEGylated liposomes (GSH-PEG-L). Endocytosis and exocytosis of liposomes including the role of secreted extracellular vesicles (EVs) of brain endothelial cells (BECs) were assessed. Furthermore, pharmacokinetics and brain distribution analysis of gemcitabine loaded liposomes were carried out in healthy rats to ascertain the in vivo applicability. Our findings suggested that the presence of GSH increased the cellular uptake of liposomes by up to 3-fold in human brain microvascular endothelial cells depending on the dose but not in astrocytes. The cell exposure to liposomes particularly GSH-PEG-L dramatically increased the cell secretion of small and microvesicles with liposomal components, though different liposomes preferred different vesicles for exocytosis. This correlated with GSH-PEG-L transport efficiency of 4 % across the in vitro BBB model in 24 h, 1.7-fold higher than that of PEG-L (p < 0.05). In rats, while PEG-L and GSH-PEG-L showed similar pharmacokinetic profiles and prolonged circulation properties, 3.8 % of the total injected dose (ID) of gemcitabine was found in the brain of the GSH-PEG-L group at 8 h post-injection, compared with 2.8 % ID in the PEG-L group. A brain: blood concentration ratio of 1.27 ± 0.12 indicated that an active transport mechanism to cross the BBB for GSH-PEG-L. Overall, this study revealed that GSH augmented the transcellular transport efficiency of liposomes through BBB to improve targeted brain delivery by enhancing cellular uptake and vesicular exocytosis route of BECs.

The Gelatinase Inhibitor ACT-03 Reduces Gliosis in the Rapid Kindling Rat Model of Epilepsy, and Attenuates Inflammation and Loss of Barrier Integrity In Vitro.

Matrix metalloproteinases (MMPs) are endopeptidases responsible for the cleavage of intra- and extracellular proteins. Several brain MMPs have been implicated in neurological disorders including epilepsy. We recently showed that the novel gelatinase inhibitor ACT-03 has disease-modifying effects in models of epilepsy. Here, we studied its effects on neuroinflammation and blood–brain barrier (BBB) integrity. Using the rapid kindling rat model of epilepsy, we examined whether ACT-03 affected astro- and microgliosis in the brain using immunohistochemistry. Cellular and molecular alterations were further studied in vitro using human fetal astrocyte and brain endothelial cell (hCMEC/D3) cultures, with a focus on neuroinflammatory markers as well as on barrier permeability using an endothelial and astrocyte co-culture model. We observed less astro- and microgliosis in the brains of kindled animals treated with ACT-03 compared to control vehicle-treated animals. In vitro, ACT-03 treatment attenuated stimulation-induced mRNA expression of several pro-inflammatory factors in human fetal astrocytes and brain endothelial cells, as well as a loss of barrier integrity in endothelial and astrocyte co-cultures. Since ACT-03 has disease-modifying effects in epilepsy models, possibly via limiting gliosis, inflammation, and barrier integrity loss, it is of interest to further evaluate its effects in a clinical trial.