Tesmilifene modifies brain endothelial functions and opens the blood-brain/blood-glioma barrier.

Tesmilifene, a tamoxifen analog with antihistamine action, has chemopotentiating properties in experimental and clinical cancer studies. In our previous works, tesmilifene increased the permeability of the blood-brain barrier (BBB) in animal and culture models. Our aim was to investigate the effects of tesmilifene on brain microvessel permeability in the rat RG2 glioma model and to reveal its mode of action in brain endothelial cells. Tesmilifene significantly increased fluorescein extravasation in the glioma. Short-term treatment with tesmilifene reduced the resistance and increased the permeability for marker molecules in a rat triple co-culture BBB model. Tesmilifene also affected the barrier integrity in brain endothelial cells co-cultured with RG2 glioblastoma cells. Tesmilifene inhibited the activity of P-glycoprotein and multidrug resistance-associated protein-1 efflux pumps and down-regulated the mRNA expression of tight junction proteins, efflux pumps, solute carriers, and metabolic enzymes important for BBB functions. Among the possible signaling pathways that regulate BBB permeability, tesmilifene activated the early nuclear translocation of NFκB. The MAPK/ERK and PI3K/Akt kinase pathways were also involved. We demonstrate for the first time that tesmilifene increases permeability marker molecule extravasation in glioma and inhibits efflux pump activity in brain endothelial cells, which may have therapeutic relevance. Tesmilifene, a chemopotentiator in experimental and clinical cancer studies increases vascular permeability in RG2 glioma in rats and permeability for marker molecules in a culture model of the blood-brain barrier. Tesmilifene inhibits the activity of efflux pumps and down-regulates the mRNA expression of tight junction proteins, transporters, and metabolic enzymes important for the blood-brain barrier functions, which may have therapeutic relevance.

Efflux transport of serum amyloid P component at the blood-brain barrier.

Serum amyloid P component (SAP), a member of the innate immune system, does not penetrate the brain in physiological conditions; however, SAP is a stabilizing component of the amyloid plaques in neurodegenerative diseases. We investigated the cerebrovascular transport of human SAP in animal experiments and in culture blood-brain barrier (BBB) models. After intravenous injection, no SAP could be detected by immunohistochemistry or ELISA in healthy rat brains. Salmonella typhimurium lipopolysaccharide injection increased BBB permeability for SAP and the number of cerebral vessels labeled with fluorescein isothiocyanate (FITC)-SAP in mice. Furthermore, when SAP was injected to the rat hippocampus, a time-dependent decrease in brain concentration was seen demonstrating a rapid SAP efflux transport in vivo. A temperature-dependent bidirectional transport of FITC-SAP was observed in rat brain endothelial monolayers. The permeability coefficient for FITC-SAP was significantly higher in abluminal to luminal (brain to blood) than in the opposite direction. The luminal release of FITC-SAP from loaded endothelial cells was also significantly higher than the abluminal one. Our data indicate the presence of BBB efflux transport mechanisms protecting the brain from SAP penetration. Damaged BBB integrity due to pathological insults may increase brain SAP concentration contributing to development of neurodegenerative diseases.

Protection of the blood-brain barrier by pentosan against amyloid-ß-induced toxicity.

Endothelial cells of brain capillaries forming the blood-brain barrier play an important role in the pathogenesis and therapy of Alzheimer's disease. Amyloid-ß (Aß) peptides are key pathological elements in the development of the disease. A blood-brain barrier model, based on primary rat brain endothelial cells was used in which the barrier properties were induced by glial cells. The effects of amyloid peptides have been tested on cell viability and barrier functions. Aß showed toxic effects on primary rat brain endothelial cells measured by MTT dye conversion and the lactate dehydrogenase release. Morphologically cytoplasmic vacuolization, disruption of the structure of cytoplasmic organelles and tight junctions could be observed in brain endothelial cells. Treatment with Aß1-42 decreased the electrical resistance, and increased the permeability of brain endothelial cell monolayers for both fluorescein and albumin. Serum amyloid P component which stabilizes Aß fibrils in cortical amyloid plaques and cerebrovascular amyloid deposits significantly potentiated the barrier-weakening effect of Aß1-42. Sulfated polysaccharide pentosan could decrease the toxic effects of Aß peptides in brain endothelial cells. It could also significantly protect the barrier integrity of monolayers from damaging actions of peptides. Pentosan modified the size, and significantly decreased the number of amyloid aggregates demonstrated by atomic force microscopy. The present data further support the toxic effects of amyloid peptides on brain endothelial cells, and can contribute to the development of molecules protecting the blood-brain barrier in Alzheimer's disease.

Abnormalities of pituitary function after traumatic brain injury in children.

Traumatic brain injury (TBI) is a frequent cause of neuroendocrine dysfunction typically in male adults. Head injuries are also common in childhood, but only a few case reports outlined the endocrine consequences. The aim of this study was to reveal anterior pituitary function in children with history of hospitalization due to mild to severe head trauma. Our endocrine follow-up study was performed between October 2003 and February 2004 in the Pediatric Department of Petz Aladár County Teaching Hospital, Gyor, Hungary. Twenty-six children (17 boys and nine girls, aged 11.47 +/- 0.75 years) at 30.6 +/- 8.3 months after head injury and 21 age-matched controls were enrolled. Basal and stimulated anterior pituitary and peripheral hormone concentrations were measured by routine laboratory methods. Pituitary dysfunction was detected in 61% of patients with TBI history. All growth hormone (GH) parameters measured and calculated were significantly (p < 0.05) lower in TBI group than in controls after L-DOPA stimulation. Similar difference was detected 60 min after insulin provocation. Forty-two percent of all TBI children showed insufficient growth hormone (GH) response in both stimulation tests, 73% of these cases were boys. Cortisol levels of TBI patients were significantly (p < 0.05) lower all through the insulin test than values measured in control group. The degree of pituitary dysfunction was independent from the severity of TBI. Our study confirms the high risk for hypopituitarism in children with TBI despite the lack of obvious clinical symptoms. We suggest screening of pituitary function after any kind of brain trauma requiring hospitalization in childhood.

Pentosan polysulfate protects brain endothelial cells against bacterial lipopolysaccharide-induced damages.

Peripheral inflammation can aggravate local brain inflammation and neuronal death. The blood-brain barrier (BBB) is a key player in the event. On a relevant in vitro model of primary rat brain endothelial cells co-cultured with primary rat astroglia cells lipopolysaccharide (LPS)-induced changes in several BBB functions have been investigated. LPS-treatment resulted in a dose- and time-dependent decrease in the integrity of endothelial monolayers: transendothelial electrical resistance dropped, while flux of permeability markers fluorescein and albumin significantly increased. Immunostaining for junctional proteins ZO-1, claudin-5 and beta-catenin was significantly weaker in LPS-treated endothelial cells than in control monolayers. LPS also reduced the intensity and changed the pattern of ZO-1 immunostaining in freshly isolated rat brain microvessels. The activity of P-glycoprotein, an important efflux pump at the BBB, was also inhibited by LPS. At the same time production of reactive oxygen species and nitric oxide was increased in brain endothelial cells treated with LPS. Pentosan polysulfate, a polyanionic polysaccharide could reduce the deleterious effects of LPS on BBB permeability, and P-glycoprotein activity. LPS-stimulated increase in the production of reactive oxygen species and nitric oxide was also decreased by pentosan treatment. The protective effect of pentosan for brain endothelium can be of therapeutical significance in bacterial infections affecting the BBB.

Permeability studies on in vitro blood-brain barrier models: physiology, pathology, and pharmacology.

(1) The specifically regulated restrictive permeability barrier to cells and molecules is the most important feature of the blood-brain barrier (BBB). The aim of this review was to summarize permeability data obtained on in vitro BBB models by measurement of transendothelial electrical resistance and by calculation of permeability coefficients for paracellular or transendothelial tracers. (2) Results from primary cultures of cerebral microvascular endothelial cells or immortalized cell lines from bovine, human, porcine, and rodent origin are presented. Effects of coculture with astroglia, neurons, mesenchymal cells, blood cells, and conditioned media, as well as physiological influence of serum components, hormones, growth factors, lipids, and lipoproteins on the barrier function are discussed. (3) BBB permeability results gained on in vitro models of pathological conditions including hypoxia and reoxygenation, neurodegenerative diseases, or bacterial and viral infections have been reviewed. Effects of cytokines, vasoactive mediators, and other pathogenic factors on barrier integrity are also detailed. (4) Pharmacological treatments modulating intracellular cyclic nucleotide or calcium levels, and activity of protein kinases, protein tyrosine phosphatases, phospholipases, cyclooxygenases, or lipoxygenases able to change BBB integrity are outlined. Barrier regulation by drugs involved in the metabolism of nitric oxide and reactive oxygen species, as well as influence of miscellaneous treatments are also listed and evaluated. (5) Though recent advances resulted in development of improved in vitro BBB model systems to investigate disease modeling, drug screening, and testing vectors targeting the brain, there is a need for checking validity of permeability models and cautious interpretation of data.

Human serum amyloid P component attenuates the bacterial lipopolysaccharide-induced increase in blood-brain barrier permeability in mice.

Endotoxin challenge leads to septic shock, multi-organ failure and death in mice. Permeability of the blood-brain barrier (BBB) is increased by endotoxemia. Serum amyloid P component (SAP) is a lipopolysaccharide (LPS)-binding protein that can modulate the host reactions during infections. It is controversial whether SAP can protect from LPS toxicity in vivo or not. We have tested the effect of human SAP on BBB permeability of Salmonella typhimurium LPS-injected mice. The animals showed signs of sickness behaviour including immobility, anorexia, and diarrhoea. Intraperitoneally administered LPS increased the BBB permeability for sodium fluorescein for about 4-fold, and for albumin for more than 2-fold in brain cortex. SAP, given intravenously, had no effect on basal BBB permeability for albumin, although it decreased sodium fluorescein extravasation to brain tissue. In LPS-treated mice, SAP administration alleviated the symptoms of septic shock, and significantly inhibited the enhanced BBB permeability for both tracers. Our data indicate that human SAP may counteract the toxic effects of LPS during septic shock.

Adrenomedullin, an autocrine mediator of blood-brain barrier function.

Since the discovery that adrenomedullin gene expression is 20- to 40-fold higher in endothelial cells than even in the adrenal medulla, this peptide has been regarded as an important secretory product of the vascular endothelium, together with nitric oxide, eicosanoids, endothelin-1, and other vasoactive metabolites. Cerebral endothelial cells secrete an exceptionally large amount of adrenomedullin, and the adrenomedullin concentration is about 50% higher in the cerebral circulation than in the peripheral vasculature. The adrenomedullin production of cerebral endothelial cells is induced by astrocyte-derived factors. Adrenomedullin causes vasodilation in the cerebral circulation, may participate in the maintenance of the resting cerebral blood flow, and may be protective against ischemic brain injury. Recent data from our laboratory indicate that adrenomedullin, as an endothelium-derived autocrine/paracrine hormone, plays an important role in the regulation of specific blood-brain barrier properties. Adrenomedullin is suggested to be one of the physiological links between astrocyte-derived factors, cyclic adenosine 3'5'-monophosphate (cAMP), and the induction and maintenance of the blood-brain barrier. Moreover, the role of adrenomedullin in the differentiation and proliferation of endothelial cells and in angiogenesis suggests a more complex function for adrenomedullin in the cerebral circulation and in the development of the blood-brain barrier.

Transient forebrain ischemia increases the blood-brain barrier permeability for albumin in stroke-prone spontaneously hypertensive rats.

1. The aim of the present study was to reveal the effect of transient forebrain ischemia on the regional and temporal changes in the permeability of the blood-brain barrier (BBB) permeability for sodium fluorescein (MW: 376 Da) and Evan's blue-labeled albumin (MW: 67 kDa) in stroke-prone spontaneously hypertensive rats (SHRSP). 2. BBB permeability was significantly higher in the brain regions of 16-week-old control SHRSP than those in age-matched normotensive Wistar-Kyoto rats. 3. Transient forebrain ischemia evoked by 10-min bilateral carotid occlusion increased the permeability of the BBB for albumin, but not for sodium fluorescein, after 6 and 24 h of reperfusion in brain regions of SHRSP. 4. Extravasation of serum macromolecules may contribute to neuronal loss and development of hypertensive encephalopathy in SHRSP.