Peripheral nerve pericytes originating from the blood-nerve barrier expresses tight junctional molecules and transporters as barrier-forming cells

The objective of this study was to establish pure blood-nerve barrier (BNB)-derived peripheral nerve pericyte cell lines and to investigate their unique properties as barrier-forming cells. We isolated peripheral nerve, brain, and lung pericytes from transgenic rats harboring the temperature-sensitive simian virus 40 large T-antigen gene. These cell lines expressed several pericyte markers such as alpha-smooth muscle actin, NG2, osteopontin, and desmin, whereas they did not express endothelial cell markers such as vWF and PECAM. In addition, these cell lines expressed several tight junction molecules such as occludin, claudin-12, ZO-1, and ZO-2. In particular, the expression of occludin was detected in peripheral nerve and brain pericytes, although it was not detected in lung pericytes by a Western blot analysis. An immunocytochemical analysis confirmed that occludin and ZO-1 were localized at the cell-cell boundaries among the pericytes. Brain and peripheral nerve pericytes also showed significantly higher trans-pericyte electrical resistance values and lower inulin clearances than lung pericytes. We considered that occludin localized at the cell-cell boundaries among the pericytes might mechanically stabilize the microvessels of the BNB and the blood-brain barrier. Furthermore, we also showed that these cell lines expressed many barrier-related transporters. ABCG2, p-gp, MRP-1, and Glut-1 were detected by a Western blot analysis and were observed in the cytoplasm and outer membrane by an immunocytochemical analysis. These transporters on pericytes might facilitate the peripheral nerve-to-blood efflux and blood-to-peripheral nerve influx transport of substrates in cooperation with those on endothelial cells in order to maintain peripheral nerve homeostasis.     

Infection of human pericytes by HIV‐1 disrupts the integrity of the blood–brain barrier

Human immunodeficiency virus type 1 (HIV‐1) infection of the central nervous system (CNS) affects cross‐talk between the individual cell types of the neurovascular unit, which then contributes to disruption of the blood–brain barrier (BBB) and the development of neurological dysfunctions. Although the toxicity of HIV‐1 on neurons, astrocytes and brain endothelial cells has been widely studied, there are no reports addressing the influence of HIV‐1 on pericytes. Therefore, the purpose of this study was to evaluate whether or not pericytes can be infected with HIV‐1 and how such an infection affects the barrier function of brain endothelial cells. Our results indicate that human brain pericytes express the major HIV‐1 receptor CD4 and co‐receptors CXCR4 and CCR5. We also determined that HIV‐1 can replicate, although at a low level, in human brain pericytes as detected by HIV‐1 p24 ELISA. Pericytes were susceptible to infection with both the X4‐tropic NL4‐3 and R5‐tropic JR‐CSF HIV‐1 strains. Moreover, HIV‐1 infection of pericytes resulted in compromised integrity of an in vitro model of the BBB. These findings indicate that human brain pericytes can be infected with HIV‐1 and suggest that infected pericytes are involved in the progression of HIV‐1‐induced CNS damage.     

Loss and recovery of the blood–nerve barrier in the rat sciatic nerve after crush injury are associated with expression of intercellular junctional proteins

The blood–nerve barrier in peripheral nerves is important for maintaining the environment for axons. Breakdown of the barrier by nerve injury causes various pathologies. We hypothesized that the breakdown and recovery of the blood–nerve barrier after injury are associated with the changes in the expression of intercellular junctional proteins. To test this hypothesis, we induced crush injuries in the rat sciatic nerve by ligation and analyzed spatiotemporal changes of claudin-1, claudin-5, occludin, VE-cadherin, and connexin43 by immunoconfocal microscopy and morphometry and compared them with changes in the permeability of the blood–nerve barrier by intravenous and local administration of Evans blue–albumin (EBA). On day 1 after removal of the ligature EBA leaked into the connective tissue in the endoneurium and then the leakage gradually decreased and disappeared on day 7. On day 1 claudin-1, claudin-5, occludin, VE-cadherin, and connexin43 had totally disappeared from the perineurium and endoneurium. Thereafter, claudin-1, claudin-5, occludin, and VE-cadherin recovered from day 2, whereas connexin43 was redetected on day 5. These results indicate that the breakdown and following recovery of the blood–nerve barrier are closely associated with changes in the expression of claudins, occludin, VE-cadherin, and connexin43 and that the recovery time course is similar but nonidentical.     

Control of the blood–brain barrier function in cancer cell metastasis

Cerebral metastases are the most common brain neoplasms seen clinically in the adults and comprise more than half of all brain tumours. Actual treatment options for brain metastases that include surgical resection, radiotherapy and chemotherapy are rarely curative, although palliative treatment improves survival and life quality of patients carrying brain‐metastatic tumours. Chemotherapy in particular has also shown limited or no activity in brain metastasis of most tumour types. Many chemotherapeutic agents used systemically do not cross the blood–brain barrier (BBB), whereas others may transiently weaken the BBB and allow extravasation of tumour cells from the circulation into the brain parenchyma. Increasing evidence points out that the interaction between the BBB and tumour cells plays a key role for implantation and growth of brain metastases in the central nervous system. The BBB, as the tightest endothelial barrier, prevents both early detection and treatment by creating a privileged microenvironment. Therefore, as observed in several in vivo studies, precise targetting the BBB by a specific transient opening of the structure making it permeable for therapeutic compounds, might potentially help to overcome this difficult clinical problem. Moreover, a better understanding of the molecular features of the BBB, its interrelation with metastatic tumour cells and the elucidation of cellular mechanisms responsible for establishing cerebral metastasis must be clearly outlined in order to promote treatment modalities that particularly involve chemotherapy. This in turn would substantially expand the survival and quality of life of patients with brain metastasis, and potentially increase the remission rate. Therefore, the focus of this review is to summarise the current knowledge on the role and function of the BBB in cancer metastasis.     

Characteristics of glycine transport across the inner blood–retinal barrier

Although glycine plays a pivotal role in neurotransmission and neuromodulation in the retina and is present in high concentration in the retina, the source of retinal glycine is still unclear. The purpose of the present study was to investigate glycine transport across the inner blood–retinal barrier (inner BRB). [¹⁴C]Glycine transport at the inner BRB was characterized using a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2 cells) as an in vitro model of the inner BRB and in vivo vascular injection techniques. [¹⁴C]Glycine uptake by TR-iBRB2 cells was Na⁺- and Cl⁻-dependent, and concentration-dependent with Michaelis–Menten constants of 55.4 μM and 8.02 mM, and inhibited by glycine transporter 1 (GlyT1) and system A inhibitors. These uptake studies suggest that GlyT1 and system A are involved in [¹⁴C]glycine uptake by TR-iBRB2 cells. RT-PCR analysis demonstrated that GlyT1 and system A (encoding ATA 1 and ATA2) mRNA are expressed in TR-iBRB2 cells. An in vivo study suggested that [¹⁴C]glycine is transported from blood to the retina whereas [¹⁴C]α-methylaminoisobutyric acid, a selective substrate for system A, is not. In conclusion, GlyT1 most likely mediates glycine transport at the inner BRB and is expected to play an important role in regulating the glycine concentration in the neural retina.     

Hyperammonemia enhances the function and expression of P‐glycoprotein and Mrp2 at the blood–brain barrier through NF‐κB

Ammonia is considered to be the main neurotoxin responsible for hepatic encephalopathy resulting from liver failure. Liver failure has been reported to alter expression and activity of P‐glycoprotein (P‐gp) and multidrug resistance‐associated protein 2 (Mrp2) at the blood–brain barrier (BBB). The aim of this study was to investigate whether ammonia is involved in abnormalities of expression and activity of P‐gp and Mrp2 at the BBB. Hyperammonemic rats were developed by an intraperitoneal injection of ammonium acetate (NH₄Ac, 4.5 mmol/kg). Results showed that Mrp2 function markedly increased in cortex and hippocampus of rats at 6 h following NH₄Ac administration. Significant increase in function of P‐gp was observed in hippocampus of rats. Meanwhile, such alterations were in line with the increase in mRNA and protein levels of P‐gp and Mrp2. Significant increase in levels of nuclear amount of nuclear factor‐κB (NF‐κB) p65 was also observed. Primarily cultured rat brain microvessel endothelial cells (rBMECs) were used for in vitro study. Data indicated that 24 h exposure to ammonia significantly increased function and expression of P‐gp and Mrp2 in rBMECs, accompanied with activation of NF‐κB. Furthermore, such alterations induced by ammonia were reversed by NF‐κB inhibitor. In conclusion, this study demonstrates that hyperammonemia increases the function and expression of P‐gp and Mrp2 at the BBB via activating NF‐κB pathway.

     

Molecular dynamics of the blood–testis barrier components during murine spermatogenesis

The blood–testis barrier (BTB) separates the seminiferous epithelium into the adluminal and basal compartments. During murine spermatogenesis, preleptotene/leptotene spermatocytes migrate from the basal to the adluminal compartment through the BTB during stages VIII–IX. In the present study, we focused on the tight junction (TJ) molecules and analyzed their spatiotemporal expression during the murine seminiferous epithelial cycle. Structural analysis revealed that the principal components of the BTB, for example, claudin‐3, claudin‐11, occludin, and zonula occludens‐1 (ZO‐1), were localized at the basal and luminal sides of the preleptotene/leptotene spermatocytes during the migration stages (VIII–IX). Although we detected claudin‐11, occludin, and ZO‐1 throughout spermatogenesis, claudin‐3 was only detected during stages VI–IX. Quantitative PCR using dissected seminiferous tubules from three stages (Early: II–VI, Middle: VII–VIII, Late: IX–I) clarified that the mRNA levels of TJ molecules were not correlated with the histoplanimetrical protein levels during spermatogenesis. Additionally, tubulobulbar complexes, considered to be involved in the internalization of TJ, were observed at the BTB site. Furthermore, a significant reduction in the mRNA levels of genes for the degradation of occludin (Itch) and endocytic recycling (Rab13) were observed during the Late and Middle stages, respectively. Therefore, we hypothesized that the lag between mRNA and protein expression of TJ molecules may be due to posttranslational modulation, for example, tubulobulbar complexes and endocytic recycling processes. In conclusion, these findings indicate that the integrity of the BTB is maintained throughout spermatogenesis, and the stage‐specific localization of claudin‐3 protein plays an important role in regulating BTB permeability. Mol. Reprod. Dev. 77: 630–639, 2010. © 2010 Wiley‐Liss, Inc.     

Chemokine-dependent mechanisms of leukocyte trafficking across a model of the blood–brain barrier

Leukocyte transmigration across the blood–brain barrier (BBB) is a multistep process that can be mediated by chemokines. These low-molecular-weight chemoattractant proteins are secreted by cells within the central nervous system (CNS) in response to injury or on activation. Leukocytes transmigrate toward this chemokine gradient, crossing the BBB and gaining access to the CNS parenchyma. Depending on the chemokine, the nature of the insult, and the type of cell that transmigrates, the BBB integrity may be disrupted, leading to its increased permeability. Both the inflammation resulting from leukocyte transmigration and BBB perturbations contribute to CNS pathology. The mechanisms that mediate leukocyte transmigration and BBB disruption, as well as tissue culture models that are used to study leukocyte trafficking, are the focus of this review.     

A putative nicotine pump at the metabolic blood–brain barrier of the tobacco hornworm

In mammals, P-glycoprotein immunostaining at the blood–brain barrier has implicated the multidrug pump in the restricted movement of many cytotoxic agents into the central nervous system (NCS). Since many insects require as sophisticated blood–brain barrier system to protect their CNS from plant-derived neurotoxins, we have investigated the possibility that a P-glycoprotein homolog constitutes a component of the insect blood–brain barrier. We have used the nicotine-resistant tobacco hornworm (Manduca sexta) to address this issue. Manduca has been previously shown, in physiological studies, to have an alkaloid (nicotine/morphine/atropine) pump at its excretory malpighian tubules. We show (1) that the tubules are P-glycoprotein immunopositive, (2) that Manduca has a metabolic blood–brain barrier for nicotine, (3) that the barrier co-localizes with P-glycoprotein immunostaining, and (4) that detoxifying enzymes as well as the nicotine pump are likely to account for the metabolic blood–brain to nicotine. These findings may provide insights on two major fronts, the troublesome problem of multi-insecticide resistance, a phenomenon that parallels multidrug resistance in tumor cells, and the problem of tolerance to addictive neuroactive drugs like nicotine or morphine. 1994 John Wiley & Sons, Inc.     

Identification and quantification of blood–brain barrier transporters in isolated rat brain microvessels

The blood–brain barrier (BBB ) maintains brain homeostasis by tightly regulating the exchange of molecules with systemic circulation. It consists primarily of microvascular endothelial cells surrounded by astrocytic endfeet, pericytes, and microglia. Understanding the make‐up of transporters in rat BBB is essential to the translation of pharmacological and toxicological observations into humans. In this study, experimental workflows are presented in which the optimization of (a) isolation of rat brain microvessels (b) enrichment of endothelial cells, and (c) extraction and digestion of proteins were evaluated, followed by identification and quantification of BBB proteins. Optimization of microvessel isolation was indicated by 15‐fold enrichment of endothelial cell marker Glut1 mRNA , whereas markers for other cell types were not enriched. Filter‐aided sample preparation was shown to be superior to in‐solution sample preparation (10251 peptides vs. 7533 peptides). Label‐free proteomics was used to identify nearly 2000 proteins and quantify 1276 proteins in isolated microvessels. A combination of targeted and global proteomics was adopted to measure protein abundance of 6 ATP‐binding cassette and 27 solute carrier transporters. Data analysis using proprietary Progenesis and open access MaxQuant software showed overall agreement; however, Abcb9 and Slc22a8 were quantified only by MaxQuant, whereas Abcc9 and Abcd3 were quantified only by Progenesis. Agreement between targeted and untargeted quantification was demonstrated for Abcb1 (19.7 ± 1.4 vs. 17.8 ± 2.3) and Abcc4 (2.2 ± 0.7 vs. 2.1 ± 0.4), respectively. Rigorous quantification of BBB proteins, as reported in this study, should assist with translational modeling efforts involving brain disposition of xenobiotics.

     

Melatonin alleviates lipopolysaccharide‐compromised integrity of blood–brain barrier through activating AMP‐activated protein kinase in old mice

Blood–brain barrier (BBB) dysfunction is considered to be an early event in the pathogenesis of a variety of neurological diseases in old patients, and this could occur in old people even when facing common stress. However, the mechanism remains to be defined. In this study, we tested the hypothesis that decreased melatonin levels may account for the BBB disruption in old mice challenged with lipopolysaccharide (LPS), which mimicked the common stress of sepsis. Mice (24–28 months of age) received melatonin (10 mg kg^?1 day^?1, intraperitoneally, i.p.) or saline for one week before exposing to LPS (1 mg kg^?1, i.p.). Evan's blue dye (EB) and immunoglobulin G (IgG) leakage were used to assess BBB permeability. Immunostaining and Western blot were used to detect protein expression and distribution. Our results showed that LPS significantly increased BBB permeability in old mice accompanied by the degradation of tight junction proteins occludin and claudin‐5, suppressed AMP‐activated protein kinase (AMPK) activation, and elevated gp91^phox protein expression. Interestingly, administration of melatonin for one week significantly decreased LPS‐induced BBB disruption, AMPK suppression, and gp91^phox upregualtion. Moreover, activation of AMPK with metformin significantly inhibited LPS‐induced gp91^phox upregualtion in endothelial cells. Taken together, our findings demonstrate that melatonin alleviates LPS‐induced BBB disruption through activating AMPK and inhibiting gp91^phox upregulation in old mice.     

The effect of serum starvation on tight junctional proteins and barrier formation in Caco-2 cells

Assessing the ability of pharmaceutics to cross biological barriers and reach the site-of-action requires faithful representation of these barriers in vitro. Difficulties have arisen in replicating in vivo resistance in vitro. This paper investigated serum starvation as a method to increase Caco-2 barrier stability and resistance. The effect of serum starvation on tight junction production was examined using transwell models; specifically, transendothelial electrical resistance (TEER), and the expression and localization of tight junction proteins, occludin and zonula occludens-1 (ZO-1), were studied using western blotting and immunofluorescence. Changing cells to serum-free media 2 days post-seeding resulted in TEER readings of nearly 5000 Ω cm² but the TEER rapidly declined subsequently. Meanwhile, exchanging cells to serum-free media 4–6 days post-seeding produced barriers with resistance readings between 3000 and 4000 Ω cm², which could be maintained for 18 days. This corresponded to an increase in occludin levels. Serum starvation as a means of barrier formation is simple, reproducible, and cost-effective. It could feasibly be implemented in a variety of pre-clinical pharmaceutical assessments of drug permeability across various biological barriers with the view to improving the clinical translation of novel therapeutics.     

Role of Rho/ROCK signaling in the interaction of melanoma cells with the blood–brain barrier

We have investigated the role of the Rho/ROCK signaling pathway in the interaction of metastatic melanoma cells with the brain endothelium. ROCK inhibition induced a shift of melanoma cells to the mesenchymal phenotype, increased the number of melanoma cells attached to the brain endothelium, and strengthened the adhesion force between melanoma and endothelial cells. Inhibition of ROCK raised the number of melanoma cells migrating through the brain endothelial monolayer and promoted the formation of parenchymal brain metastases in vivo. We have shown that inhibition of the Rho/ROCK pathway in melanoma, but not in brain endothelial cells, is responsible for this phenomenon. Our results indicate that the mesenchymal type of tumor cell movement is primordial in the transmigration of melanoma cells through the blood–brain barrier.     

n-3 Fatty acids modulate brain glucose transport in endothelial cells of the blood–brain barrier

We have previously shown that glucose utilization and glucose transport were impaired in the brain of rats made deficient in n-3 polyunsaturated fatty acids (PUFA). The present study examines whether n-3 PUFA affect the expression of glucose transporter GLUT1 and glucose transport activity in the endothelial cells of the blood–brain barrier. GLUT1 expression in the cerebral cortex microvessels of rats fed different amounts of n-3 PUFA (low vs. adequate vs. high) was studied. In parallel, the glucose uptake was measured in primary cultures of rat brain endothelial cells (RBEC) exposed to supplemental long chain n-3 PUFA, docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids, or to arachidonic acid (AA). Western immunoblotting analysis showed that endothelial GLUT1 significantly decreased (−23%) in the n-3 PUFA-deficient microvessels compared to control ones, whereas it increased (+35%) in the microvessels of rats fed the high n-3 PUFA diet. In addition, binding of cytochalasin B indicated that the maximum binding to GLUT1 (Bmax) was reduced in deficient rats. Incubation of RBEC with 15 μM DHA induced the membrane DHA to increase at a level approaching that of cerebral microvessels isolated from rats fed the high n-3 diet. Supplementation of RBEC with DHA or EPA increased the [³H]-3-O-methylglucose uptake (reflecting the basal glucose transport) by 35% and 50%, respectively, while AA had no effect. In conclusion, we suggest that n-3 PUFA can modulate the brain glucose transport in endothelial cells of the blood–brain barrier, possibly via changes in GLUT1 protein expression and activity.     

Identification and distribution of insulin receptors on cultured bovine brain microvessel endothelial cells: Possible function in insulin processing in the blood–brain barrier

The binding of ¹²⁵ I-insulin to primary cultures of bovine brain microvessel endothlial cells was examined. Insulin binding was both time and temperature dependent and inhibited by excess unlabeled insulin. Furthermore, the specific binding of insulin was polarized to the apical side of the cell monolayers. Upon binding, the labeled insulin was internalized, with approximately 70% resistant to acid wash over a 90-min period. The inhibition of insulin internalization observed with cell monolayers exposed to either phenylarsine oxide or unlabeled insulin suggests a receptor-mediated endocytic process. Furthermore, the ability of chloroquine to reduce the metabolism of insulin indicates a significant portion of the peptide iis processed through a lysosomal pathway. In contrast to the fluid-phase endocytosis marker, Lucifer yellow, as much as 65% of internalized insulin undergoes apical to basolateral trancytosis in brain microvessel endothelial cells. While most of the effluxed insulin was degraded, as assessed by trichloroacetic acid precipitation, the results of the present study suggest insulin receptors within the brain microvasculature may be involved in the processing and transport of bloodborne insulin. © 1994 Wiley-Liss, Inc.