Very low intensity alternating current decreases cell proliferation.

Electric fields impact cellular functions by activation of ion channels or by interfering with cell membrane integrity. Ion channels can regulate cell cycle and play a role in tumorigenesis. While the cell cycle may be directly altered by ion fluxes, exposure to direct electric current of sufficient intensity may decrease tumor burden by generating chemical products, including cytotoxic molecules or heat. We report that in the absence of thermal influences, low-frequency, low-intensity, alternating current (AC) directly affects cell proliferation without a significant deleterious contribution to cell survival. These effects were observed in normal human cells and in brain and prostate neoplasms, but not in lung cancer. The effects of AC stimulation required a permissive role for GIRK2 (or K(IR)3.2) potassium channels and were mimicked by raising extracellular potassium concentrations. Cell death could be achieved at higher AC frequencies (>75 Hz) or intensities (>8.5 microA); at lower frequencies/intensities, AC stimulation did not cause apoptotic cellular changes. Our findings implicate a role for transmembrane potassium fluxes via inward rectifier channels in the regulation of cell cycle. Brain stimulators currently used for the treatment of neurological disorders may thus also be used for the treatment of brain (or other) tumors.

Significance of MDR1 and multiple drug resistance in refractory human epileptic brain.

BACKGROUND: The multiple drug resistance protein (MDR1/P-glycoprotein) is overexpressed in glia and blood-brain barrier (BBB) endothelium in drug refractory human epileptic tissue. Since various antiepileptic drugs (AEDs) can act as substrates for MDR1, the enhanced expression/function of this protein may increase their active extrusion from the brain, resulting in decreased responsiveness to AEDs. METHODS: Human drug resistant epileptic brain tissues were collected after surgical resection. Astrocyte cell cultures were established from these tissues, and commercially available normal human astrocytes were used as controls. Uptake of fluorescent doxorubicin and radioactive-labeled Phenytoin was measured in the two cell populations, and the effect of MDR1 blockers was evaluated. Frozen human epileptic brain tissue slices were double immunostained to locate MDR1 in neurons and glia. Other slices were exposed to toxic concentrations of Phenytoin to study cell viability in the presence or absence of a specific MDR1 blocker. RESULTS: MDR1 was overexpressed in blood vessels, astrocytes and neurons in human epileptic drug-resistant brain. In addition, MDR1-mediated cellular drug extrusion was increased in human 'epileptic' astrocytes compared to 'normal' ones. Concomitantly, cell viability in the presence of cytotoxic compounds was increased. CONCLUSIONS: Overexpression of MDR1 in different cell types in drug-resistant epileptic human brain leads to functional alterations, not all of which are linked to drug pharmacokinetics. In particular, the modulation of glioneuronal MDR1 function in epileptic brain in the presence of toxic concentrations of xenobiotics may constitute a novel cytoprotective mechanism.

Peripheral markers of brain damage and blood-brain barrier dysfunction.

PURPOSE: Occurrence of brain damage is frequently associated with abnormal blood-brain barrier (BBB) function. Two brain-specific proteins, S100beta and neuron-specific enolase (NSE) are released systemically in a variety of neurological diseases, but S100beta levels sometimes rise in the absence of neuronal damage, suggesting that S100beta is a marker of BBB rather than neuronal damage. METHODS: We measured both proteins in the serum of patients undergoing iatrogenic BBB disruption with intrarterial mannitol, followed by chemotherapy. RESULTS: Serum S100beta increased significantly after mannitol infusion (p<0.05) while NSE did not. Furthermore, in a model of intracerebral hemorrhage, S100beta increases in CSF did not lead to serum changes at a time when the BBB was intact. Modeling of S100beta release from the CNS suggested that low (<0.34 ng/ml) serum levels of S100beta are consistent with BBB opening without CNS damage, while larger increases imply synthesis and release from presumable damaged glia. CONCLUSIONS: Thus, S100beta in serum is an early marker of BBB openings that may precede neuronal damage and may influence therapeutic strategies. Secondary, massive elevations in S100beta are indicators of prior brain damage and bear clinical significance as predictors of poor outcome or diagnostic means to differentiate extensive damage from minor, transient impairment.

Thrombospondin-4 and its variants: expression and differential effects on endothelial cells.

BACKGROUND: In a recent large-scale genetic association study, a single nucleotide polymorphism in the thrombospondin-4 (TSP-4) gene, resulting in a proline-for-alanine substitution at position 387, was associated with a significantly increased risk for premature atherosclerosis. TSP-4 had not previously been implicated in vascular pathology, and very little information is available on its expression and functions. METHODS AND RESULTS: The goal of this study was to assess TSP-4 expression in vessel wall and to identify differences in functions of TSP-4 variants that could account for the proatherogenic effects of the (P387)TSP-4 variant. TSP-4 expression was demonstrated in human endothelial cells (ECs) and vascular smooth muscle cells from brain blood vessels and coronary arteries. (P387)TSP-4 and its fragment (residues 326 to 722), but not the A(387) forms, suppressed EC adhesion and proliferation. The (P387)TSP-4 was more active in inducing the phosphorylation of focal adhesion kinase, consistent with inhibition of proliferation. Both variant fragments increased the proliferation of human aortic smooth muscle cells. CONCLUSIONS: TSP-4 is expressed by vascular cells and influences the vessel wall by modulating the proliferation of ECs and smooth muscle cells. The A387P substitution is a "gain-of-function" mutation, favoring a form of TSP-4 that interferes with EC adhesion and proliferation and may thereby be proatherogenic.

Serum transthyretin monomer as a possible marker of blood-to-CSF barrier disruption.

The CNS is shielded from systemic influences by two separate barriers, the blood-brain barrier (BBB) and the blood-to-CSF barrier. Failure of either barrier bears profound significance in the etiology and diagnosis of several neurological diseases. Furthermore, selective opening of BBB tight junctions provides an opportunity for delivery of otherwise BBB impermeant drugs. Peripheral assessment of BBB opening can be achieved by detection in blood of brain-specific proteins that extravasate when these endothelial junctions are breached. We developed a proteomic approach to discover clusters of CNS-specific proteins with extravasation into serum that correlates with BBB openings. Protein profiles from blood samples obtained from patients undergoing iatrogenic BBB disruption (BBBD) with intra-arterial hyperosmotic mannitol were compared with pre-BBB opening serum. A low molecular weight protein (14 kDa) identified by mass spectroscopy as transthyretin (TTR) consistently correlated with BBBD. Protein gel electrophoresis and immunodetection confirmed that TTR was indeed extravasated in its monomeric form when CNS barriers were breached. The time course of TTR extravasation was compared with release from the brain of another BBB integrity marker, S-100beta (11 kDa). Kinetic analysis revealed that the appearance of S-100beta, presumably originating from perivascular astrocytic end feet, preceded extravasation of TTR by several minutes. Because TTR is localized primarily in choroid plexus and, as a soluble monomer, in CSF, we concluded that although S-100beta is a marker of BBB integrity, TTR instead may be a peripheral tracer of blood-to-cerebrospinal barrier.

A new dynamic in vitro model for the multidimensional study of astrocyte-endothelial cell interactions at the blood-brain barrier.

Blood-brain barrier endothelial cells are characterized by the presence of tight intercellular junctions, the absence of fenestrations, and a paucity of pinocytotic vesicles. The in vitro study of the BBB has progressed rapidly over the past several years as new cell culture techniques and improved technologies to monitor BBB function became available. Studies carried out on viable in vitro models are set to accelerate the design of drugs that selectively and aggressively can target the CNS. Several systems in vitro attempt to reproduce the physical and biochemical behavior of intact BBB, but most fail to reproduce the three-dimensional nature of the in vivo barrier and do not allow concomitant exposure of endothelial cells to abluminal (glia) and lumenal (flow) influences. For this purpose, we have developed a new dynamic in vitro BBB model (NDIV-BBB) designed to allow for extensive pharmacological, morphological and physiological studies. Bovine aortic endothelial cells (BAEC) developed robust growth and differentiation when co-cultured alone. In the presence of glial cells, BAEC developed elevated Trans-Endothelial Electrical Resistance (TEER). Excision of individual capillaries proportionally decreased TEER; the remaining bundles were populated with healthy cells. Flow played an essential role in EC differentiation by decreasing cell division. In conclusion, this new dynamic model of the BBB allows for longitudinal studies of the effects of flow and co-culture in a controlled and fully recyclable environment that also permits visual inspection of the abluminal compartment and manipulation of individual capillaries.