Peptides crossing the blood-brain barrier: some unusual observations

An interactive blood–brain barrier (BBB) helps regulate the passage of peptides from the periphery to the CNS and from the CNS to the periphery. Many peptides cross the BBB by simple diffusion, mainly explained by their lipophilicity and other physicochemical properties. Other peptides cross by saturable transport systems. The systems that transport peptides into or out of the CNS can be highly specific, transporting MIF-1 but not Tyr–MIF-1, PACAP38 but not PACAP27, IL-1 but not IL-2, and leptin but not the smaller ingestive peptides NPY, orexin A, orexin B, CART (55–102[Met(O)⁶⁷]), MCH, or AgRP(83–132). Although the peptides EGF and TGF-α bind to the same receptor, only EGF enters by a rapid saturable transport system, suggesting that receptors and transporters can represent different proteins. Even the polypeptide NGF enters faster than its much smaller subunit β-NGF. The saturable transport of some compounds can be upregulated, like TNF-α in EAE (an animal model of multiple sclerosis) and after spinal cord injury, emphasizing the regulatory role of the BBB. As has been shown for CRH, saturable transport from brain to blood can exert effects in the periphery. Thus, the BBB plays a dynamic role in the communication of peptides between the periphery and the CNS.     

Glucose transport to the brain: A systems model

Glucose transport to the brain involves sophisticated interactions of solutes, transporters, enzymes, and cell signaling processes, within an intricate spatial architecture. The dynamics of the transport are influenced by the adaptive nature of the blood–brain barrier (BBB), the semi-impermeable membranes of brain capillaries. As both the gate and the gatekeeper between blood-borne nutrients and brain tissue, the BBB helps govern brain homeostasis. Glucose in the blood must cross the BBB's luminal and abluminal membranes to reach neural tissue. A robust representation of the glucose transport mechanism can highlight a target for brain therapeutic intervention, help characterize mechanisms behind several disease phenotypes, or suggest a new delivery route for drugs. The challenge for researchers is understanding the relationships between influential physiological variables in vivo, and using that knowledge to predict how alterations or interventions affect glucose transport.This paper reviews factors influencing glucose transport and approaches to representing blood-to-brain glucose transport including in vitro, in vivo, and kinetic models. Applications for different models are highlighted, while their limitations in answering arising questions about the human in vivo BBB lead to a discussion of an alternate approach. A developing complex systems simulation is introduced, initiating a single platform to represent the dynamics of glucose transport across the adapting human blood–brain barrier.     

Permeability of the blood–brain barrier to neurotrophins

To evaluate the feasibility of applying blood-borne neurotrophins to promote normal function of the central nervous system (CNS) and to rescue neuronal degeneration, we characterized the permeability of the blood–brain barrier (BBB) to neurotrophins. We report here that some members of the neurotrophin family (NGF, βNGF, NT3, and NT5) can cross the BBB of mice in vivo to arrive at the brain parenchyma. BBB permeability differed among individual neurotrophins in that NGF had the fastest influx rate (K_i) and NT3 the slowest, and that the entry rate of NGF was twice that of its smaller bioactive subunit βNGF. BBB permeability also differed at various CNS regions in that the cervical spinal cord had the greatest rate of influx, whereas brain had the lowest. Saturability of influx was suggested by self-inhibition studies for NT3 in vivo, and for NGF in an in situ brain perfusion system, indicating the presence of saturable transport systems. The results suggest that peripheral administration of neurotrophins could have therapeutic effects within the CNS.     

Molecular and cellular permeability control at the blood–brain barrier

The blood–brain barrier (BBB) is formed by brain capillary endothelial cells. These cells have at least three properties which distinguish them from their peripheral counterparts: (1) tight junctions (TJs) of extremely low permeability; (2) low rates of fluid-phase endocytosis; (3) specific transport and carrier molecules. In combination, these features restrict the nonspecific flux of ions, proteins, and other substances into the central nervous system (CNS) environment. The restriction protects neurons from harmful compositional fluctuations occurring in the blood and allows uptake of essential molecules. Breakdown of the BBB is associated with a variety of CNS disorders and results in aggravation of the condition. Restoration of the BBB is thus one strategy during therapy of CNS diseases. Its success depends on a precise knowledge of the structural and functional principles underlying BBB functionality. In this review we have tried to summarise the current knowledge of TJs, including information gained from non-neuronal systems, and describe selected mechanisms involved in permeability regulation.     

Critical role of actin in modulating BBB permeability

A major obstacle in the treatment of degenerative manifestations and debilitating diseases in the central nervous system (CNS) lies in the impediment of drug delivery into these tissues. The impediment is due to a membrane barrier referred to as the blood–brain barrier (BBB). It is known that the BBB is a unique membranous structure in brain capillaries that tightly segregates the brain from systemic blood circulation. It is imperative to have a thorough understanding of the molecular components and their integrated function of this barrier to develop effective therapeutics for CNS disorders and diseases. Although there are other cell and biochemical properties that underlie this barrier function, it is well established that the barrier is mainly made up of the physical elements of tight junction (TJ) complex. The major constituents of TJ, such as occludin, claudins, zonula occludens (ZOs) and junctional adhesion molecule (JAM) have been subjects of intensive studies and reviews. However, after examining currently proposed models, we have come to believe that a cytoskeletal component-actin may play a critical role in interacting TJ molecular constituents and modulating functional TJ complex. In this review, we will discuss the correlation of temporal and spatial distribution and remodeling of actin filaments with altering integrity of TJ complexes in various systems and present a hypothesis to depict its potential role in modulating BBB permeability.     

S-adenosylmethionine is substrate for carrier mediated transport at the blood-brain barrier in vitro

S-adenosylmethionine (SAM) is the sole methyl donor in the CNS where it is involved in a multitude of biochemical reactions. Peripherally administered SAM has been shown to increase SAM levels in cerebrospinal fluid and is reported to be effective in the treatment of numerous neurological disorders suggesting SAM crosses the blood-brain barrier (BBB). The mechanism of SAM entry into the brain remains unknown, but the presence of adenosyl and methionine residues in the molecule suggests probable entry via carrier mediated transport. We have investigated whether SAM utilises endogenous transport systems in cerebral endothelial cells, using RBE4 cells, an in vitro model of the BBB. SAM did not influence the transport of [(3)H]-methionine and only marginally reduced the uptake of [(3)H]-leucine in RBE4 cells. The inhibition constant for the latter was 2.11+/-0.29 mM (mean+/-S.E.M.). However, increasing concentrations of SAM strongly inhibited the transport of [3H]-adenosine in RBE4 cells in both the presence and the absence of sodium in the medium, with K(i) values of 199+/-32 and 139+/-8.4 microM, respectively. Lineweaver-Burk plots suggest a competitive mode of inhibition. The findings suggest that SAM is not recognised by the L-system transporter for large neutral amino acids at the brain endothelium. A significant interaction with the transport of adenosine, however, indicates that SAM has affinity for the nucleoside carrier systems; this is within the range of K(m) values of natural substrates and suggest that SAM may enter the CNS via the Na(+)-independent nucleoside carrier systems at the brain capillary endothelium.     

Dendrimer Advances for the Central Nervous System Delivery of Therapeutics

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A co-culture-based model of human blood-brain barrier: application to active transport of indinavir and in vivo-in vitro correlation

The growing array of in vitro models of the blood-brain barrier (BBB) which have been used makes it difficult to draw firm conclusions concerning the BBB penetration of HIV-1 protease inhibitors. What is needed is a combined in vivo and in vitro study on biological models that mimic as closely as possible the normal human BBB, to establish whether and how indinavir crosses the BBB. We developed a new human BBB model using primary endothelial cells and astrocytes. The biological relevance of this model was checked with respect on the one hand, to the close relationship between the log of drug permeability coefficient normalized to molecular weight and the log of the 1-octanol/water partition coefficient, and on the other hand to the functional P-glycoprotein (P-gp) expression. We employed this model to perform transport studies with indinavir and showed that the rate of in vitro indinavir transport from the basal to apical compartment was higher than the rate of apical to basal transport. Pretreatment of the BBB model with the P-gp inhibitor, quinidine, significantly increased apical to basal transport. Intracellular indinavir accumulation was increased in BBB as a result of inhibition of active transport. These data were correlated with the indinavir-mediated P-gp ATPase modulation showing that indinavir specifically interacted with a binding site on P-gp. Moreover, the activation of P-gp ATPase by indinavir was inhibited by quinidine. In addition, the in vivo brain to plasma concentration ratio of indinavir into mice showed that indinavir concentration was up to five times higher in the brain of mdr1a(-/-) mice than in the brain of mdr1a(+/+) mice. All these results confirm the role of P-gp in preventing the passage of indinavir across BBB and thus its entry into the central nervous system (CNS). Our human BBB model represents a useful tool for the evaluation of drug penetration into the CNS.     

中枢神经纳米载药系统的研究进展

许多亲水性的药物,如一些抗生素、抗肿瘤药物和神经肽类在全身性给药后不能透过血脑屏障（BBB）。药物修饰、渗透性方式打开脑毛细血管内皮和选择性的给药方式（颅内给药）都是常用的提高药物进人中枢神经系统（CNS）的方法。而通过纳米载药系统,包括脂质体、固态多聚体纳米颗粒或者固体脂质纳米颗粒等,不仅可以帮助药物透过BBB,同时可以控制药物释放,延缓药物的化学及酶类降解速度。同时纳米载药系统可以降低药物对外周组织的毒性,在提高药物靶向性方面具有非常广阔的应用前景。本文简要介绍了CNS纳米载药系统的研究进展及临床应用状况。     

Electron microscope study of blood-brain barrier opening induced by immunological targeting of the endothelial barrier antigen

Barrier vessels in the central nervous system are lined with endothelial cells which constitute the blood-brain barrier (BBB) and show selective expression of certain biochemical markers. One of these, the endothelial barrier antigen (EBA), is specific to the rat. The exact role of EBA in the BBB is not known, although several studies have shown a correlation between the reduction in EBA expression in endothelial cells and the opening of the BBB. However, in these studies it was not possible to determine if EBA reduction was a primary event or was secondary to opening of the BBB. A recent light microscope study demonstrated that immunological targeting of EBA in vivo, by intravenous injection of a monoclonal antibody (anti-EBA), leads to acute and widespread opening of the BBB. In the current study we have employed this model together with tracer application and immunoperoxidase electron microscopy to determine the site of binding of the injected antibody and the route of opening of the BBB. The results showed that (a) the anti-EBA injected in vivo became bound to brain endothelial cells, principally to luminal membranes. (b) Endothelial cells showed widened intercellular junctions and increased cytoplasmic vesicles and vacuoles. (c) Many perivascular astrocytic processes were swollen. (d) The macromolecular tracer HRP was present in vesicles, vacuoles, widened paracellular clefts, the perivascular space and brain parenchyma. In conclusion, the in vivo targeting of EBA leads to opening of the BBB apparently via paracellular and transcellular routes. This model is useful for the study of vascular permeability in the CNS and experimental manipulation of the BBB. It may have a potential application in experimental studies on drug delivery throughout the CNS.     

Protein expression of brain endothelial cell E-cadherin after hypoxia/aglycemia: influence of astrocyte contact

The blood-brain barrier (BBB) plays a crucial role in protecting the central nervous system (CNS) from any changes in homeostasis brought about by pathological conditions. Cerebrovascular permeability is an important factor in the development of cerebral edema following stroke [M. Plateel, E. Teissier, R. Cecchelli, Hypoxia, dramatically increases the nonspecific transport of blood-borne proteins to the brain. J. Neurochem. 68 (1997) 874-877] and any changes in its function can have detrimental neurological consequences. Recently, research has shown that an in vitro model of the BBB is sensitive to short exposures of hypoxia/aglycemia and that changes in endothelial cell calcium flux may be responsible for structural and functional variations in the BBB during ischemic stress [T.J. Abbruscato, T.P. Davis, Combination of hypoxia/aglycemia compromises in vitro BBB. J. Pharmacol. Exp. Ther. 289 (1999) 668-675]. Present experiments investigated bovine brain microvessel endothelial cell (BBMEC) expression of a Ca(2+)-dependent cell-cell adhesion molecule, E-cadherin, which has been shown to be important for blood-brain barrier function [D. Pal, K.L. Audus, T.J. Siahaan, Modulation of cellular adhesion in bovine brain microvessel endothelial cells by a decapeptide. Brain Research 747 (1997) 103-113]. Since it is believed that astrocyte-endothelial cell interaction is crucial for maintenance of in vivo BBB characteristics, we have attempted to optimize our isolation and culturing techniques to produce a reliable, in vitro model of the BBB that is suitable to study pathological conditions. Immunofluoresence experiments showed positive staining for E-cadherin, yet failed to show any change in cellular distribution of E-cadherin upon hypoxic/aglycemic exposure. In addition, culturing BBMECs with C6 conditioned medium (CM) had no effect on the localization of E-cadherin. Western blotting experiments showed that BBMECs express E-cadherin and this protein is decreased in a time dependent manner after various hypoxic/aglycemic exposures when endothelial cells are cultured alone or with C6 astrogliomas grown on a separate culture surface. When C6 astrocytes are grown directly opposed to endothelial cells, with a porous membrane between, we observed a slight attenuation in the decreased BBMEC expression of E-Cadherin after hypoxia/aglycemia exposure. This work has shown that the mammalian brain endothelial/astrocyte co-culture system is a useful model for studies of pathological conditions where BBB characteristics are maintained.     

Tyrosine phosphatase inhibition induces loss of blood-brain barrier integrity by matrix metalloproteinase-dependent and -independent pathways

Tight junctions between endothelial cells of brain capillaries form the structural basis of the blood-brain barrier (BBB), which controls the exchange of molecules between blood and CNS. Regulation of cellular barrier permeability is a vital and complex process involving intracellular signalling and rearrangement of tight junction proteins. We have analysed the impact of tyrosine phosphatase inhibition on tight junction proteins and endothelial barrier integrity in a primary cell culture model based on porcine brain capillary endothelial cells (PBCEC) that closely mimics the BBB in vitro. The tyrosine phosphatase inhibitor phenylarsine oxide (PAO) induced increased matrix metalloproteinase (MMP) activity, which was paralleled by severe disruption of cell-cell contacts and proteolysis of the tight junction protein occludin. ZO-1 and claudin-5 were not affected. Under these conditions, the transendothelial electrical resistance (TEER) was markedly reduced. PAO-induced occludin proteolysis could be prevented by different MMP inhibitors. Pervanadate (PV) reduced the TEER similar to PAO, but did not increase MMP activity. Cell-cell contacts of PV-treated cells appeared unaffected, and occludin proteolysis did not occur. Our results suggest that tyrosine phosphatase inhibition can influence barrier properties independent of, but also correlated to MMPs. Evidence is given for a role of MMPs in endothelial tight junction regulation at the BBB in particular and probably at tight junctions (TJs) in general.     

The critical component to establish in vitro BBB model: Pericyte

The blood–brain barrier (BBB), a highly regulated membranous barrier of brain capillaries, consists of an intricate network of tight junctions (TJs) that segregate the central nervous system (CNS) from systemic blood circulation and maintain a delicate homeostasis of the CNS environment. While endothelial cells (ECs) of brain capillaries are clearly the principal cellular element of BBB, the formation and regulation of intact BBB structure appear to require the interactions of endothelial cells with other cellular components. Astrocytes, one of the major non-neural cells in the brain, associate closely and interact with capillary endothelial cells during the angiogenesis and BBB development. Current in vitro cellular models for the study of BBB functions often incorporate astrocytes with endothelial cells. However, another foremost cell type, CNS pericyte, which intimately embraces brain capillary endothelium, attracts relatively little attention for its role in developing the in vitro BBB system. This review will analyze the critical functions of pericytes in angiogenesis in various systems and discuss the relevance of these functions in mediating the development, maintenance, and regulation of BBB. The author will also discuss the functional role of actin in both ECs and pericytes, and further elaborate the molecular mechanisms of BBB permeability regulation that involves the transduction pathway-mediated actin remodeling process. Finally, the rationale of incorporating pericytes for establishing a better in vitro BBB model will be emphasized.     

Blood–brain barrier and traumatic brain injury

The blood–brain barrier (BBB) is an anatomical microstructural unit, with several different components playing key roles in normal brain physiological regulation. Formed by tightly connected cerebrovascular endothelial cells, its normal function depends on paracrine interactions between endothelium and closely related glia, with several recent reports stressing the need to consider the entire gliovascular unit in order to explain the underlying cellular and molecular mechanisms. Despite that, with regard to traumatic brain injury (TBI) and significant events in incidence and potential clinical consequences in pediatric and adult ages, little is known about the actual role of BBB disruption in its diverse pathological pathways. This Mini‐Review addresses the current literature on possible factors affecting gliovascular units and contributing to posttraumatic BBB dysfunction, including neuroinflammation and disturbed transport mechanisms along with altered permeability and consequent posttraumatic edema. Key mechanisms and its components are described, and promising lines of basic and clinical research are identified, because further knowledge on BBB pathological interference should play a key role in understanding TBI and provide a basis for possible therapeutic targets in the near future, whether through restoration of normal BBB function after injury or delivering drugs in an increased permeability context, preventing secondary damage and improving functional outcome. © 2013 Wiley Periodicals, Inc.     

Drug transport across the blood–brain barrier

The blood–brain barrier (BBB) prevents the brain uptake of most pharmaceuticals. This property arises from the epithelial-like tight junctions within the brain capillary endothelium. The BBB is anatomically and functionally distinct from the blood–cerebrospinal fluid barrier at the choroid plexus. Certain small molecule drugs may cross the BBB via lipid-mediated free diffusion, providing the drug has a molecular weight <400 Da and forms <8 hydrogen bonds. These chemical properties are lacking in the majority of small molecule drugs, and all large molecule drugs. Nevertheless, drugs can be reengineered for BBB transport, based on the knowledge of the endogenous transport systems within the BBB. Small molecule drugs can be synthesized that access carrier-mediated transport (CMT) systems within the BBB. Large molecule drugs can be reengineered with molecular Trojan horse delivery systems to access receptor-mediated transport (RMT) systems within the BBB. Peptide and antisense radiopharmaceuticals are made brain-penetrating with the combined use of RMT-based delivery systems and avidin–biotin technology. Knowledge on the endogenous CMT and RMT systems expressed at the BBB enable new solutions to the problem of BBB drug transport.     

Molecular anatomy of intercellular junctions in brain endothelial and epithelial barriers: electron microscopist’s view

In this review, we have tried to summarize the current knowledge on the distribution of important molecular components of intercellular junctions—both tight junctions (TJs) and adherens junctions (AJs)—at the level of ultrastructure. For this purpose, immunogold procedure was applied to ultrathin sections of brain samples obtained from mice, rats, and humans and embedded in hydrophilic resin Lowicryl K4M. The results of our observations performed with transmission electron microscopy (EM) are discussed and compared with findings of other authors. Although the main structures responsible for the barrier and fence functions of the blood–brain barrier (BBB) and blood–CSF barrier are TJs present between endothelial cells (ECs) of brain capillaries and epithelial cells of the choroid plexus, their functional characteristics (e.g. tightness of the barrier evaluated by electrical resistance) differ significantly. Therefore, our main attention is focused on the presence and distribution of both intrinsic, i.e. integral membrane (transmembrane), molecules such as occludin, claudins, and junctional adhesion molecule (JAM) in TJs, and cadherins in AJs, as well as peripheral molecules of both types of junctions, e.g. zonula occludens (ZO) proteins and catenins. The latter group of molecules connects transmembrane proteins with the cell cytoskeleton. A close spatial association of the TJ proteins with those of AJs indicates that both junctional types are intermingled in the BBB type of endothelium. One of most important purposes of this work is to find out the junction-associated molecules that can serve as sensitive markers of normal or disturbed function of brain barriers. Understanding the structural–functional relations between molecular components of junctional complexes in physiological and experimental conditions of both barriers can provide important information about the etiology of various pathological conditions of the central nervous system and also help to elaborate new therapeutic approaches.     

Transmission routes of HIV-1 gp120 from brain to lymphoid tissues

The blood-brain barrier (BBB) restricts the entry of antiviral agents into the CNS thereby facilitating the creation of a reservoir of HIV that could potentially reinfect peripheral tissues. We characterized the efflux from brain of radioactively labeled viral coat HIV-1 gp120 (I-gp120) after intracerebroventricular (i.c.v.) injection. The half-time disappearance rate of I-gp120 from brain was 12.6 min, which was faster than could be explained by the reabsorption of cerebrospinal fluid into blood but could not be explained by a saturable transporter. After i.c.v. injection, I-gp120 appeared in the serum and was sequestered by spleen and the cervical nodes, demonstrating a potential for virus within the CNS to reinfect peripheral tissues. However, the amount of I-gp120 appearing in serum was less than that expected based on the efflux rate, whereas uptake by the cervical nodes was much greater after i. c.v. than after i.v. injection of I-gp120. These findings were explained by drainage from the brain directly to the cervical lymph nodes through the brain's primitive lymphatic system. These lymphatics potentially provide a pathway through which CNS reservoirs of HIV-1 could directly reinfect lymphoid tissue without being exposed to circulating antiviral agents.     

Persistence of blood-to-brain transport of leptin in obese leptin-deficient and leptin receptor-deficient mice

In lean CD-1 mice, leptin is delivered into the brain by a saturable transport mechanism. Previous work has shown that obesity is associated with decreased leptin transport. Here, we investigated the transport of leptin across the blood–brain barrier (BBB) in two murine models of obesity. Radioiodinated leptin was intravenously injected into ob/ob (no leptin production) and db/db (high leptin levels, but no long-form leptin receptor) mutant mice and their lean controls. In all groups, the labeled polypeptide was transported across the BBB by a saturable mechanism. The rates of transport were not significantly different between the mutant strains and their lean controls. The results demonstrate that leptin transport persists in the absence of production of the endogenous polypeptide or its signal-transducing receptor and suggest that the impaired transport previously seen is not directly explained by only obesity or alterations in serum plasma levels.     

Interleukin-2 does not cross the blood-brain barrier by a saturable transport system

Blood-borne interleukin-2 (IL-2), like other cytokines, is known to affect the central nervous system (CNS). One mechanism by which circulating substances can alter brain function is to directly cross the blood-brain barrier (BBB). We investigated the ability of IL-2 to cross the BBB, the interface between the periphery and the CNS. IL-2 labeled with ¹²⁵I (I-IL-2) was injected into mice intravenously and its rate of entry into the brain determined by multiple-time regression analysis. I-IL-2 was found to enter the brain about 10 times faster than albumin. Neither morphine nor antibodies to IL-2, IL-1α, or the IL-1 receptor affected the entry of I-IL-2. High performance liquid chromatography (HPLC) confirmed that the radioactivity entering the brain represented intact cytokine. However, excess unlabeled IL-2 was unable to impede the entry of I-IL-2, indicating that this transport is nonsaturable. This contrasts with saturable transport systems found for the cytokines IL-1α and TNF-α, but still may explain how IL-2 can exert central effects.     

Hematopoietic growth factors pass through the blood-brain barrier in intact rats

We have previously demonstrated that receptors for hematopoietic growth factors, stem cell factor (SCF) and granulocyte-colony stimulating factor (G-CSF) are expressed in the neurons and the neural progenitor cells (NPCs) of the adult rat brain, and that systemic administration of SCF and G-CSF in the first week after induction of cortical brain ischemia (3 h–7 days post-ischemia) significantly improves functional outcome, augments NPC proliferation, and reduces infarct volume in rats. The purpose of the present study is to determine whether SCF and G-CSF pass through the blood–brain barrier (BBB) in intact rats. The growth factors were labeled with iodine (I¹²⁵), a radioactive compound. I¹²⁵-SCF and I¹²⁵-G-CSF were intravenously administered and the concentrations of I¹²⁵-SCF and I¹²⁵-G-CSF in the blood plasma and the brain were determined at 10, 30, 60, and 120 min after injection. We observed that both SCF and G-CSF were slowly and continuously transported from the blood stream to the brain in the same rate. In addition, both immunofluorescent staining and Western blots showed that receptors for SCF and G-CSF were expressed in the capillaries of the adult rat brain, suggesting that SCF and G-CSF entry to the brain may be mediated via receptor-mediated transport, one of the endogenous transports in the BBB. These data indicate that both SCF and G-CSF were able to pass through the BBB in intact animals. This observation will help in further exploring the physiological role of peripheral SCF and G-CSF in the brain and therapeutic possibility to chronic stroke.