Rachel C. Brown

Calcium Modulation of Adherens and Tight Junction Function: A Potential Mechanism for Blood-Brain Barrier Disruption After Stroke

Background— This review deals with the role of calcium in endothelial cell junctions of the blood-brain barrier (BBB). Calcium is critical for adherens junction function, but it appears that calcium is also important in regulating tight junction function necessary for the barrier characteristics of cerebral microvessels. Summary of Review— The BBB is critical for brain homeostasis and is located at the cerebral microvessel endothelial cells. These endothelial cells maintain their barrier characteristics via cell-cell contacts made up of adherens and tight junctions. Adherens junctions are calcium dependent; recent evidence suggests that calcium also affects tight junctions. After stroke, there is a disruption of the BBB. Interfering with calcium flux under hypoxic conditions can prevent BBB breakdown. Calcium may alter BBB junction integrity by a number of different signal transduction cascades, as well as via direct interaction of calcium ions with junction proteins. It remains to be determined whether clinical use of calcium channel antagonists is a viable means to reduce BBB disruption after stroke. Conclusions— With the widespread use of calcium channel blockers as clinical treatments for hypertension, which is a risk factor for stroke, the exact role of calcium in modulating BBB integrity needs to be elucidated.

Tight junction protein expression and barrier properties of immortalized mouse brain microvessel endothelial cells

Understanding the molecular and biochemical mechanisms regulating the blood-brain barrier is aided by in vitro model systems. Many studies have used primary cultures of brain microvessel endothelial cells for this purpose. However, primary cultures limit the generation of material for molecular and biochemical assays since cells grow slowly, are prone to contamination by other neurovascular unit cells, and lose blood-brain barrier characteristics when passaged. To address these issues, immortalized cell lines have been generated. In these studies, we assessed the suitability of the immortalized mouse brain endothelial cell line, bEnd3, as a blood-brain barrier model. RT-PCR and immunofluorescence indicated expression of multiple tight junction proteins. bEnd3 cells formed barriers to radiolabeled sucrose, and responded like primary cultures to disrupting stimuli. Exposing cells to serum-free media on their basolateral side significantly decreased paracellular permeability; astrocyte-conditioned media did not enhance barrier properties. The serum-free media-induced decrease in permeability was correlated with an increase in claudin-5 and zonula occludens-1 immunofluorescence at cell-cell contracts. We conclude that bEnd3 cells are an attractive candidate as a model of the blood-brain barrier due to their rapid growth, maintenance of blood-brain barrier characteristics over repeated passages, formation of functional barriers and amenability to numerous molecular interventions.

Nicotine increases in vivo blood-brain barrier permeability and alters cerebral microvascular tight junction protein distribution.

The blood-brain barrier (BBB) is critical to the health of the central nervous system. The BBB is formed primarily by the presence of tight junctions (TJ) between cerebral microvessel endothelial cells. In light of the known effects of nicotine on endothelial cell biology, the specific effects of nicotine on the in vivo BBB were examined. Using in situ brain perfusion, it was found that continuous administration of nicotine (4.5 mg free base x kg(-1) x day(-1)) for 1 and 7 days led to increased permeability of the BBB to [14C]-sucrose without significant changes in its initial volume of distribution. The expression and distribution of the TJ-associated proteins actin, occludin, claudin-1, -3, and -5, and ZO-1 and -2 were analyzed by Western blot and immunofluorescence microscopy. Though no changes in total protein expression were observed, nicotine treatment was associated with altered cellular distribution of ZO-1 and diminished junctional immunoreactivity of claudin-3. It is proposed that nicotine leads to changes in BBB permeability via the modulation of TJ proteins.

Protection against hypoxia-induced increase in blood-brain barrier permeability: Role of tight junction proteins and NFκB

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 NFκB.

Smoking and ischemic stroke: a role for nicotine?

Cigarette smoking is a preventable risk factor for ischemic stroke. The mechanisms by which smoking contributes to stroke are poorly understood and the role of nicotine in this process is controversial. Although nicotine administered transdermally and orally does not appear to have as many associated health risks as do cigarettes, nicotine does have acute vasoactive and mitogenic effects on vascular tissues. Nicotine might alter the function of the blood-brain barrier and disrupt normal endothelial cell function. Some of the detrimental effects of nicotine are prevented by nicotinic acetylcholine receptor antagonists. However, recent studies indicate that nicotine might also interact with intracellular signaling pathways that are independent of acetylcholine receptors. In light of these recent developments, the impact of nicotine on cerebrovascular pathology should not be dismissed.

Mannitol opening of the blood–brain barrier: regional variation in the permeability of sucrose, but not 86Rb+ or albumin

Clinically, infusion of hyperosmolar solutions is used to enhance chemotherapeutic drug penetration of the blood-brain barrier (BBB) in patients with malignant brain tumors or metastases. We examined the effect of hyperosmolar BBB disruption on brain permeability of three compounds, 86Rb+, a marker for K+ permeability and transport, [14C]sucrose and Evans blue albumin, using a rat in situ perfusion model. 86Rb+ and [14C]sucrose had increased permeability 20 min after BBB disruption with 1.6 M mannitol. There was no change in Evans blue albumin permeability. Only [14C]sucrose showed regional variation in permeability after mannitol-induced BBB disruption, with the cortex and midbrain having higher sucrose permeability then either the cerebellum or brainstem. These data suggest that the clinical efficacy of hyperosmolar disruption therapy in conjunction with chemotherapeutic agents, of a similar molecular weight to sucrose, may be affected by the location of the tumor within the brain.

TRPC-mediated actin-myosin contraction is critical for BBB disruption following hypoxic stress

Hypoxia-induced disruption of the blood-brain barrier (BBB) is the result of many different mechanisms, including alterations to the cytoskeleton. In this study, we identified actin-binding proteins involved in cytoskeletal dynamics with quantitative proteomics and assessed changes in subcellular localization of two proteins involved in actin polymerization [vasodilator-stimulated phosphoprotein (VASP)] and cytoskeleton-plasma membrane cross-linking (moesin). We found significant redistribution of both VASP and moesin to the cytoskeletal and membrane fractions of BBB endothelial cells after 1-h hypoxic stress. We also investigated activation of actin-myosin contraction through assessment of phosphorylated myosin light chain (pMLC) with confocal microscopy. Hypoxia caused a rapid and transient increase in pMLC. Blocking MLC phosphorylation through inhibition of myosin light chain kinase (MLCK) with ML-7 prevented hypoxia-induced BBB disruption and relocalization of the tight junction protein ZO-1. Finally, we implicate the transient receptor potential (TRP)C family of channels in mediating these events since blockade of TRPC channels and the associated calcium influx with SKF-96365 prevents hypoxia-induced permeability changes and the phosphorylation of MLC needed for actin-myosin contraction. These data suggest that hypoxic stress triggers alterations to cytoskeletal structure that contribute to BBB disruption and that calcium influx through TRPC channels contributes to these events.

Regulation of Blood‐Brain Barrier Permeability by Transient Receptor Potential Type C and Type V Calcium‐Permeable Channels

Objective: To identify plasma membrane ion channels mediating calcium influx at the blood‐brain barrier in response to disrupting stimuli.

Viability of microvascular endothelial cells to direct exposure of formalin, λ-carrageenan, and complete Freund's adjuvant

We investigated three inflammatory agents to establish if these substances elicit a direct effect on the functional and structural integrity of the blood-brain barrier. Cellular cytotoxicity and paracellular permeability were assessed in vitro using primary bovine brain microvascular endothelial cells exposed to formalin, lambda-carrageenan, or complete Freunds adjuvant for 1, 3, or 72 h, respectively. Results showed that only the highest concentration (0.025%) of formalin produced a decrease in cell viability (approximately 34%) and a significant increase in cell permeability to [(14)C]sucrose at 120 min (approximately 137%). Brain perfusion using female Sprague-Dawley rats showed no difference in paracellular permeability to [(14)C]sucrose for any inflammatory agent. Western blot analyses were performed on isolated rat brain microvessels to assess the structural integrity of blood-brain barrier tight junctions. Results indicate that expression of zonula occludens-1, occludin, claudin-1, and actin remain unchanged following intravenous exposure to inflammatory agents. This study confirms that changes seen at the blood-brain barrier following a peripheral inflammation are due to physiological responses to the given inflammatory agent and not to any direct interaction between the inflammatory agent and the brain microvasculature.

Mechanosensitive Calcium Fluxes in the Neurovascular Unit: TRP Channel Regulation of the Blood-Brain Barrier

The blood-brain barrier of the neurovascular unit is a critical organ for normal brain function. It is continuously exposed to mechanical stress both from the peripheral circulation and plasma osmolarity changes. Barrier integrity is regulated by a number of mechanisms, including influx of calcium ions and activation of calcium-sensitive signaling pathways. This review addresses the molecular identity of the channels underlying mechanosensitive calcium influx, and hypothesizes a central role for transient receptor potential channels in mechanosensitive regulation of blood-brain barrier endothelial cell function.