Brain Microvessel Endothelial Cells Research Articles

Protection against hypoxia-induced blood-brain barrier disruption: changes in intracellular calcium.

Tissue damage after stroke is partly due to disruption of the blood-brain barrier (BBB). Little is known about the role of calcium in modulating BBB disruption. We investigated the effect of hypoxic and aglycemic stress on BBB function and intracellular calcium levels. Bovine brain microvessel endothelial cells were treated with A-23187 to increase intracellular calcium without hypoxia or treated with a calcium chelator (BAPTA) or calcium channel blockers (nifedipine or SKF-96365) and 6 h of hypoxia. A-23187 alone did not increase paracellular permeability. Hypoxia increased intracellular calcium, and hypoxia or hypoxia-aglycemia increased paracellular permeability. Treatment with nifedipine and SKF-96365 increased intracellular calcium under normoglycemic conditions, instead of blocking calcium influx, and was protective against hypoxia-induced BBB disruption under normoglycemia. Protection by nifedipine and SKF-96365 was not due to antioxidant properties of these compounds. These data indicate that increased intracellular calcium alone is not enough to disrupt the BBB. However, increased intracellular calcium after drug treatment and hypoxia suggests a potential mechanism for these drugs in BBB protection; nifedipine and SKF-96365 plus hypoxic stress may trigger calcium-mediated signaling cascades, altering BBB integrity.

Induction of β-chemokine secretion by human brain microvessel endothelial cells via CD40/CD40L interactions

β-chemokines play an important role during the course of central nervous system (CNS) inflammation. Using primary cultures of human cerebral microvascular endothelial cells, we detected increased monocyte chemoattractant protein-1 (MCP-1) and regulated upon activation normal T cell expressed and secreted (RANTES) production following incubation with soluble CD40L. These results suggest a potential mechanism by which activated CD40L positive T cells may enhance β-chemokine expression and thus influence the recruitment of mononuclear cells across the human blood–brain barrier.

Uptake Studies for Evaluating Activity of Efflux Transporters in a Cell Line Representative of the Blood‐Brain Barrier

In this unit, protocols are presented for performing uptake studies with bovine brain microvessel endothelial cells (BBMECs) using either radiolabeled compounds, or rhodamine 123 as a marker for a P-glycoprotein substrate. The protocols can easily be modified for the investigation of substrate uptake in other cell lines.

Specific role of polysorbate 80 coating on the targeting of nanoparticles to the brain

It was reported that nanoparticles with polysorbate 80 (Tween 80, T-80) coating represented tools used for delivering drugs to brain. Nevertheless, disputations were once aroused for some complications. Aimed to have a better understanding of the specific role of T-80 coating on nanoparticles and simplify the problem, the direct observation of brain targeting combined with in vivo experiments was carried out in this work using the model nanoparticles (MNPs). The presence of a complex composed by the model loading, T-80 and nanoparticles was found in the preparation of MNPs. The result was further supported by some surface properties of MNPs. Being bound to nanoparticles that were overcoated by T-80 later, was necessary for the loading to be delivered to brain. Partial coverage was enough for T-80 coating to play a specific role in brain targeting. It seemed that brain targeting of nanoparticles was concerned with the interaction between T-80 coating and brain micro-vessel endothelial cells. Therefore, the specific role of T-80 coating on nanoparticles in brain targeting was confirmed.

The Protective Effects of Preconditioning on Cerebral Endothelial Cells in Vitro

Ischemic preconditioning (PC) can markedly reduce ensuing ischemic damage. Although most attention has focused on the neuronal effects of PC, the authors have recently shown that ischemic PC reduces ischemia-induced cerebrovascular damage. In vivo, it is difficult to ascertain whether this is a direct cerebrovascular effect of PC. This study, therefore, examined whether cerebral endothelial cells can be preconditioned in vitro in the absence of other cell types. Experiments were performed on an immortalized mouse brain endothelial cell line or primary cultures of mouse brain microvessel endothelial cells. Cells were exposed to oxygen glucose deprivation (OGD) of either short duration, as a PC stimulus, or a long duration (5 hours) with or without reoxygenation to induce endothelial damage. Endothelial injury was assessed by measuring lactate dehydrogenase release and the expression of intercellular adhesion molecule-1 at the protein and mRNA levels. Experiments indicated that 1 hour of OGD was the optimal PC stimuli and that a 1 or 3 day interval was the optimal time interval between the PC stimulus and the injurious event. Preconditioned cells had less lactate dehydrogenase release during OGD (+/- reoxygenation) and reduced intercellular adhesion molecule-1 expression after OGD with reoxygenation. This study shows that cerebral endothelial cells can be directly preconditioned. The importance of this phenomenon in the overall effects of PC on the brain remains to be elucidated. Understanding the protective mechanisms elicited by PC may give insight into how to prevent ischemia-induced vascular damage (e.g., hemorrhagic transformation).

Central nervous functions of insulin

Systemic insulin has rapid access to the brain by a receptor-mediated transport system located in endothelial cells of brain microvessels. In several species, including man, parallel increases in the concentration of insulin in the cerebrospinal fluid (CSF) after acute elevation of plasma insulin levels are well documented. In animal studies central nervous insulin has been shown to play an important role in body weight regulation and memory function. Direct intracerebroventricular administration of insulin reduced food intake and caused weight loss. Moreover, insulin in the brain has been shown to improve memory function. However, in humans the investigation of central nervous effects of insulin has been hampered by the fact, that infusion of insulin and glucose is not practicable for a longer time to obtain changes in body weight. Recently, we provided evidence, that insulin enters the CSF compartment after intranasal administration without absorption into the blood stream. With this approach, we investigated the effect of long-term intranasal administration on body weight regulation in healthy subjects. After a baseline of 2 weeks 42 lean subjects (16 females, 26 males) were treated for 8 weeks with intranasal insulin (4×40 IU/day) or placebo in a double blind, between subject comparison. Body weight and body composition were measured weekly by bioimpedance analysis. Blood glucose and plasma insulin levels were identical during intranasal administration of insulin and placebo, indicating that no relevant amount of insulin entered the blood stream. There was no decrease in body weight and body fat mass during intranasal insulin administration in woman as compared to placebo. However, in contrast to women, insulin treated men continuously lost body weight compared to the placebo condition (baseline adjusted body weight at the end of the study, placebo: 80.11±0.54kg; insulin: 78.39±0.56kg, p<0.05). Most important, this weight loss was mainly a result of a reduction in body fat mass (placebo: 14.1±0.58kg, insulin: 12.6±0.58kg, p<0.05). With the same approach, effects of intranasal administration of insulin (4×40 IU/day) or placebo for 1 week on memory function were investigated in 32 healthy subjects (16 females, 16 males). Subjects were presented with a word list containing 30 words. One week later they had to tell the words they remembered. Compared to the baseline condition intranasal administration of insulin significantly improved memory function (words recalled, placebo: 3.67±1.34, insulin: 7.52±1.38, p<0.05). There was no difference in memory function in males and females. These results indicate an important role for insulin in body weight regulation and memory function in humans. Concerning recent findings of a reduced transport of insulin across the blood brain barrier in obese subjects and in Alzheimer patients, these findings could be of considerable clinical relevance.

Nitric oxide mediates hypoxia-induced changes in paracellular permeability of cerebral microvasculature.

Ischemic stroke from a reduction in blood flow to the brain microvasculature results in a subsequent decreased delivery of oxygen (i.e., hypoxia) and vital nutrients to endothelial, neuronal, and glial cells. Hypoxia associated with stroke has been shown to increase paracellular permeability of the blood-brain barrier, leading to the release of cellular mediators and brain tissue injury. Whereas reperfusion does not occur in all ischemic strokes, increased permeability has been seen in posthypoxic reoxygenation. Currently, it is unknown whether these deleterious effects result from cellular mechanisms stimulated by decreased oxygen during stroke or posthypoxic reoxygenation stress. This study used primary bovine brain microvessel endothelial cells (BBMECs) to examine the involvement of nitric oxide (NO) as a mediator in hypoxia-induced permeability changes. Hypoxia-induced increased transport of [14C]sucrose across BBMEC monolayers compared with normoxia was attenuated by either posthypoxic reoxygenation or inhibition of NO synthase (NOS). The hypoxia-induced permeability effect was further reduced when NOS inhibition was combined with posthypoxic reoxygenation. Additionally, a significant increase in total NO was seen in BBMECs after hypoxic exposure. This correlation was supported by the increased [14C]sucrose permeability observed when BBMECs were exposed to the NO donor diethylenetriaamine NONOate. Western blot analyses of NOS isoforms showed a significant increase in the inducible isoform after hypoxic exposure with a subsequent reduction in expression on reoxygenation. Results from this study suggest that hypoxia-induced blood-brain barrier breakdown can be diminished by inhibition of NO synthesis, decreased concentration of NO metabolites, and/or reoxygenation.

Release of prostaglandin E-2 in bovine brain endothelial cells after exposure to three unique forms of the antifungal drug amphotericin-B: role of COX-2 in amphotericin-B induced fever

Common formulations of amphotericin-B include a deoxycholate colloidal suspension (d-Amph), an amphotericin-B lipid complex (Ablc), and a liposomal product (l-Amph). The clinical incidence of infusion related fever is highest with d-Amph, intermediate with Ablc, and lowest with l-Amph. In the present study, we measured the activation of cyclooxygenase-2 (COX-2) and subsequent release of prostaglandin E-2 (PgE-2) from brain microvessel endothelium treated with these three formulations of amphotericin-B. Primary cultured bovine brain microvessel endothelial cells (BBMEC) were exposed to d-Amph, Ablc and l-Amph at concentrations that can be achieved in the plasma of patients receiving the drug. Media samples from the cells were collected and analyzed for PgE-2. Release of PgE-2 from BBMEC monolayers treated with l-Amph was similar to cells receiving culture media alone. In contrast, Ablc and d-Amph caused significantly greater release of PgE-2 from BBMEC monolayers compared to controls receiving culture media alone. PgE-2 release after d-Amph treatment was similar in magnitude to that observed with bacterial lipopolysaccharide (LPS). Western blot analysis indicated significant induction of COX-2 expression in BBMEC following LPS, Ablc or d-Amph treatment. Furthermore, PgE-2 release following exposure of BBMEC monolayers to either LPS or the various amphotericin-B formulations was reduced by the addition of the selective COX-2 inhibitor, NS-398. These studies indicate that amphotericin-B induces COX-2 expression in brain microvessel endothelium resulting in release of fever producing PgE-2. The magnitude of PgE-2 release from BBMEC following exposure to various amphotericin-B formulations mirrors the clinical observations regarding amphotericin-B induced fever and serves as initial support for the clinical use of COX-2 inhibitors to reduce amphotericin-B fever.

Corticotropin-releasing factor (CRF) can directly affect brain microvessel endothelial cells

Stress activates the hypothalamic–pituitary–adrenal (HPA) axis through release of corticotropin releasing factor (CRF), leading to production of glucocorticoids that down regulate immune responses. However, acute stress via CRF also has pro-inflammatory effects. We previously showed that acute stress increases rat blood–brain barrier (BBB) permeability, an effect involving brain mast cells and CRF, as it was absent in W/W v mast cell-deficient mice and was blocked by the CRF-receptor antagonist, Antalarmin. We investigated if CRF could also have a direct action on brain microvessel endothelial cells (BMEC) isolated from rat and bovine brain. BMEC were cultured and identified by electron microscopy. Western blot analysis of cultured BMEC identified CRF receptor protein; stimulation with CRF, or it structural analogue urocortin (Ucn) showed that the receptor is functionally coupled to adenylate cyclase as it increased cyclic AMP (cAMP) levels by 2-fold. These findings suggest that CRF could affect BMEC structure or function, as reported for increased cAMP levels by other studies. It is, therefore, possible that CRF may directly regulate BBB permeability, in addition to any effect mediated via brain mast cells.

Optimal structure requirements for pluronic block copolymers in modifying P-glycoprotein drug efflux transporter activity in bovine brain microvessel endothelial cells.

Pluronic block copolymer P85 was shown to inhibit the P-glycoprotein (Pgp) drug efflux system and to increase the permeability of a broad spectrum of drugs in the blood-brain barrier (BBB). However, there is an entire series of Pluronics varying in lengths of propylene oxide and ethylene oxide and overall lipophilicity. This study identifies those structural characteristics of Pluronics required for maximal impact on drug efflux transporter activity in bovine brain microvessel endothelial cells (BBMECs). Using a wide range of block copolymers, differing in hydrophilic-lipophilic balance (HLB), this study shows that lipophilic Pluronics with intermediate length of propylene oxide block (from 30 to 60 units) and HLB <20 are the most effective at inhibiting Pgp efflux in BBMECs. The methods used included 1) cellular accumulation studies with the Pgp substrate rhodamine 123 in BBMECs to assess Pgp activity; 2) luciferin/luciferase ATP assay to evaluate changes in cellular ATP; 3) 1,6-diphenyl-1,3,5-hexatriene membrane microviscosity studies to determine alterations in membrane fluidity; and 4) Pgp ATPase assays using human Pgp-expressing membranes. Pluronics with intermediate lipophilic properties showed the strongest fluidization effect on the cell membranes along with the most efficient reduction of intracellular ATP synthesis in BBMEC monolayers. The relationship between the structure of Pluronic block copolymers and their biological response-modifying effects in BBMECs are useful for determining formulations with maximal efficacy for increasing BBB permeability.

Zonula Occludens Toxin Increases the Permeability of Molecular Weight Markers and Chemotherapeutic Agents Across the Bovine Brain Microvessel Endothelial Cells

The purpose of this study was to examine the ability of Zonula occludens toxin (Zot) to reversibly open tight junctions in bovine brain microvessel endothelial cells (BBMECs) to enhance drug delivery via the paracellular pathway. Transport across BBMEC monolayers was examined for molecular weight markers and chemotherapeutic agents ([(14)C]sucrose, [(14)C]inulin, [(3)H]propranolol, [(3)H]doxorubicin, and [(14)C]paclitaxel) with Zot (0.0-4.0 microg/mL). TEER of monolayers was measured to assess effect and reversibility of Zot. Cell viability of BBMEC in the presence of Zot was assessed by trypan blue exclusion staining. Apparent permeability (P(app)), enhancement ratio (R), and percent increase in transport determined were statistically compared by ANOVA. A significant increase (p < 0.05) in P(app) was observed for the transport of [(14)C]sucrose, [(14)C]inulin, [(3)H]doxorubicin, and [(14)C]paclitaxel at a 4.0 microg/mL concentration of Zot. A significant concentration-dependent decrease in TEER was observed on treatment with Zot with rapid reversal to baseline after removal. Zot (4 micro/ml) was found to be nontoxic to the BBMECs after 2 hours incubation. In conclusion, Zot increased paracellular transport across the BBMEC in a reversible, concentration-dependent manner. Modulation of paracellular transport with Zot may be used to increase the brain permeability of potent central nervous system-active drugs, including anticancer agents.

Computation of log BB values for compounds transported through carrier-mediated mechanisms using in vitro permeability data from brain microvessel endothelial cell (BMEC) monolayers.

To explore the possibility of determining in vivo log BB values (the logarithm value of brain to plasma concentration ratio) from in vitro permeability data measured in brain microvessel endothelial cell (BMEC) monolayers. An equilibrium mathematical model was developed: log BB = log(Ca/Cb) + log Kbr:pl, where Cb and Ca are the drug concentrations at equilibrium in the basolateral (B) and apical (A) sides of BMECs in an A-to-B directional diffusion system and Kbr:pl is the brain-plasma partition coefficient. With this model, murine log BB values were calculated for 24 pharmaceutical compounds, mostly Pgp substrates. Calculated log BB values correlated well to experimental values (r2 = 0.854, slope = 0.907 +/- 0.080), demonstrating that the model could reasonably predict brain penetration for compounds that are involved in carrier-mediated transport mechanisms. For a second data set that included volatile organic compounds (log BB = log Kbr:pl), log Kbr:pl values were also shown to correlate well with their respective experimental log BB values (r2 = 0.876, slope = 0.973 +/- 0.082), demonstrating that log Kbr:pl is an excellent descriptor for log BB when a compound penetrates the blood-brain barrier by passive diffusion only. The equilibrium model demonstrated a reasonable ability to compute in vivo log BB values, regardless of the involvement or mechanisms of carrier-mediated transport.

Development of In Vitro Blood-Brain Barrier Model with Bovine Brain Microvessel Endothelial Cells

Development of In Vitro Blood-Brain Barrier Model with Bovine Brain Microvessel Endothelial Cells

Human brain microvessel endothelial cell and leukocyte interactions.

Human brain microvessel endothelial cell and leukocyte interactions.

Protection against hypoxia-induced increase in blood-brain barrier permeability: role of tight junction proteins and NFkappaB.

Co-culture with glial cells and glia-conditioned media can induce blood-brain barrier properties in microvessel endothelial cells and protect against hypoxia-induced blood-brain barrier breakdown. We examined the effect of two types of glia-conditioned media on brain microvessel endothelial cell permeability and tight junction protein expression, and studied potential mechanisms of action. We found that C6-glioma-conditioned media, but not rat astrocyte-conditioned media, protected against an increase in permeability induced by exposure to 1% oxygen for 24 hours. This hypoxic stress caused an increase in the expression of tight junction proteins claudin-1 and actin, particularly in cells treated with C6-conditioned media. We found that C6-conditioned media has a significantly higher level of both basic fibroblast growth factor and vascular endothelial growth factor. Treatment with C6-conditioned media for 1 or 3 days protects against hypoxia-induced permeability increases, and this protective effect may be mediated by signal transduction pathways terminating at the transcription factor NFkappaB.

CD40 expressed by human brain endothelial cells regulates CD4+ T cell adhesion to endothelium

Recent evidence suggests that interactions between CD40 on antigen presenting cells (APC) and CD40L on T cells generate signals that result in the activation of APC. In this study, the expression and function of CD40 was investigated in primary cultures of human brain microvessel endothelial cells (HBMEC). Results revealed constitutive expression of CD40 on untreated HBMEC. Stimulation with TNF-α, IFN-γ, LPS or combination of TNF-α and IFN-γ significantly upregulated CD40. The majority of CD40 molecules were localized on the apical surface of EC. Incubation of HBMEC with soluble CD40L resulted in increased expression of the adhesion molecules E-selectin, VCAM-1 and ICAM-1. Consequently, the adhesion of both resting and anti-CD3 activated CD4+ T lymphocytes to CD40L treated HBMEC was significantly increased compared to unstimulated EC. The expression of CD40 by cerebral endothelium, and endothelial cell activation following binding of CD40 to its ligand, CD40L, suggest a potential mechanism by which activated CD40L expressing T cells could enhance adhesion and migration of inflammatory cells across the blood–brain barrier (BBB) to sites of inflammation in the human central nervous system (CNS).

Effect of a single high-dose gamma irradiation on cultured cells in human cerebral arteriovenous malformation

Object. The purpose of this study was to analyze the effect of single high-dose gamma irradiation at a cellular biological level on tissue cultures obtained in patients who underwent surgery for cerebral arteriovenous malformation (AVM). Methods. The cell proliferation indices and changes in activation of p53, p21Waf-1, and mdm-2 were determined. Additionally, immunohistochemical investigations for vimentin, desmin, α—smooth muscle actin (α-SMA), glial fibrillary acidic protein, Factor VIII—related antigen (F-VIII), cytokeratin, S100, and transforming growth factor—β (TGFβ) were performed on cultured AVM cells after a single high-dose irradiation. Normal human brain microvessel endothelial (HBE) cells and aortic smooth muscle cells served as controls. The proliferation index decreased on the 5th day after irradiation and remained depressed over the observation period in the irradiated AVM cultures. The p53, p21Waf-1, and mdm-2 messenger RNA measurements showed considerable elevation both in AVM cultures and HBE cells after 15-Gy irradiation, which indicated apoptosis. Immunohistochemistry revealed strong vimentin positivity in the nonirradiated cultures, which gradually decreased in the irradiated cultures. Transforming growth factor—β positivity was demonstrated in the irradiated specimens, indicating transformation of fibroblastic cells into activated myofibroblastic elements. This transformation was confirmed by demonstrating elevated SMA expression as well in the radiation-treated fibroblasts. Conclusions. The presence of TGFβ and α-SMA activity in the irradiated AVM cells suggests that along with the genetically confirmed apoptotic activity, fibroblast transformation into myofibroblasts might be one of the mechanisms leading to shrinkage and obliteration of AVMs after single high-dose gamma irradiation.

Nicotine and Cotinine Modulate Cerebral Microvascular Permeability and Protein Expression of ZO-1 through Nicotinic Acetylcholine Receptors Expressed on Brain Endothelial Cells

The blood-brain barrier (BBB) adapts to a variety of pathological processes. Little is known about the effects of nicotine exposure on BBB function and the ability to adapt to stroke conditions. We have demonstrated, using a well-characterized in vitro BBB model, bovine brain microvessel endothelial cells (BBMEC) model, that nicotine and its major metabolite, cotinine, modulate BBB integrity by opening the paracellular route of solute entry into the brain. Additionally, nicotine and cotinine together increase the permeability change observed after 6 h of hypoxia/aglycemia, an in vitro model of stroke. This has important implications for how the BBB initially adapts to stroke in an environment that is previously exposed to nicotine. Nicotine and cotinine exposure also resulted in reduced ZO-1 immunoreactivity (tight junctional protein) that occurred in a time-dependent manner. Interestingly, attenuation of bovine brain microvessel endothelial cell (BBMEC) ZO-1 protein expression was reversed using 10 nM BGT, an alpha7 nicotinic acetycholine receptor (nAChR) antagonist, suggesting that the effects of nicotine on BBMEC protein expression of ZO-1 protein are mediated by nAChR expressed on brain endothelial cells. In addition to alpha7, we found that BBMEC also contain positive immunoreactivity for the alpha3, alpha5, beta2, beta3 nAChR subunit. Both alpha7 and beta2 nAChR subunit protein levels decreased with prior nicotine and cotinine exposure. These data provide evidence that nicotine and cotinine alter BBB permeability and tight junctional protein expression of ZO-1, thereby altering the BBB response to stroke conditions. These changes in brain endothelial cell paracellular permeability are believed to be associated with nicotine binding to nAChRs present at the BBB.

An in vitro Model for Blood Brain Barrier Permeation

The ability to permeate accross the blood brain barrier (BBB) is essential for drugs acting on the central nervous system (CNS). Thus, systems that allow rapid and inexpensive screening of the BBB-permeability properties of novel lead compounds are of great importance for speeding up the drug discovery process in the CNS-area. We used immortalized porcine brain microvessel endothelial cells (PBMECICl-2) to develop a model for measurement of blood-brain barrier permeation of CNS active drugs. Investigation of different cell culture conditions showed, that a system using C6 astrocyte glioma conditioned medium and addition of a cyclic AMP analog in combination with a type IV phosphodiesterase inhibitor (R020-1724) leads to cell layers with transendothelial electrical resistance values up to 300 Ω.cm2. Permeability studies with U-[14C]sucroseg ave a permeability coefficient Pe of 3.24 + 0.14 × 10−4 cm/min, which is in good agreement to published values and thus indicates the formation of tight junctions in vitro.

Comparative Anatomy, Physiology and In Vitro Models of the Blood-Brain and Blood-Retina Barrier

The blood-brain barrier (BBB) represents a functional interface between the blood stream and the neuronal microenvironment. Distinct cellular and molecular features of brain microvessel endothelial cells result in barrier and carrier functions that guarantee exclusion of adverse components such as neurotoxic metabolites on the one hand and selective passage of essential nutrients on the other hand. Circumventricular organs are brain structures that lack BBB characteristics, allowing for hormone-mediated interactions between e.g. the pituitary and distant organs. The retina, as an integral part of the central nervous system, is enclosed by the pigment epithelium, which functions as a barrier interface between the systemic blood vessels of the neighboring choroid and the retina. Various BBB-specific markers, tight junction components, and carrier systems including amino acid and saccharide transporters have been cloned. P-glycoprotein has been of special interest because this efflux pump counteracts entry of numerous therapeutically relevant drugs into the nervous system. Various in vitro systems of the retinal pigment epithelium (RPE) have been established and employed to analyze pharmacological aspects and pathological cell interactions. The most advanced systems are organotypic cultures and acute preparations of the RPE, i.e. fully intact tissue sheets that can be used as in vivo-like BBB models for transport studies and drug profiling.