Danica B. Stanimirovic

The expression and functional characterization of ABCG2 in brain endothelial cells and vessels.

Delivery of drugs to the brain is impeded by the activity of efflux pumps expressed by endothelial cells of brain vasculature. The ATP binding cassette (ABC) transporters, among which ABCB1/MDR1 P‐glycoprotein and ABCC1/multidrug resistance‐associated protein 1 are expressed in brain endothelial cells, participate in drug efflux properties of the blood‐brain barrier (BBB). Searches of the EST (expressed sequence tags) database with the conserved ABC domain, conducted to identify other ABC transporters expressed in the BBB, recovered 15 ABC transporter sequences expressed in human brain cDNA libraries. One of these sequences, identical to ABCG2, was highly expressed in cultured human cerebromicrovascular endothelial cells and human brain tissue at both mRNA and protein levels. Overexpression of human ABCG2 in immortalized rat brain endothelial cells resulted in enhanced polarized abluminal to luminal transport of various substrates tested in the in vitro BBB model. Brain vessels extracted from tissue sections of nonmalignant human brain and glioblastoma tomors by laser capture microdissection microscopy and analyzed by real‐time polymerase chain reaction showed higher expression of ABCG2 relative to ABCB1/MDR1 and ABCC1/MRP1. ABCG2 was up‐regulated in both glioblastoma vessels and parenchymal tissue. These studies suggest a role for brain endothelial ABCG2 transporter in modulating drug delivery to the brain and in conferring drug resistance to glioblastomas.

Endothelin induction of adhesion molecule expression on human brain microvascular endothelial cells.

The adhesion of circulating leukocytes to vascular endothelium is a prerequisite for their emigration to extravascular tissues. The experiments presented here demonstrate that intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) are constitutively expressed on cerebromicrovascular endothelial cell lines derived from human brain and that the expression of these molecules can be up-regulated by endothelins (ET-1, ET-2, and ET-3) in a dose- and time-dependent manner. The data also indicate that ET-1 treatment induced the expression of E-selectin on these cells. These findings implicate vasoactive peptides in the recruitment of blood cells at sites of inflammation.

AMPA receptor-mediated regulation of a Gi-protein in cortical neurons

Excitatory synaptic transmission in the central nervous system is mediated primarily by the release of glutamate from presynaptic terminals onto postsynaptic channels gated by N -methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors,. The myriad intracellular responses arising from the activation of the NMDA and AMPA receptors have previously been attributed to the flow of Ca2+ and/or Na + through these ion channels. Here we report that the binding of the agonist AMPA to its receptor can generate intracellular signals that are independent of Ca2+ and Na+ in rat cortical neurons. In the absence of intracellular Ca2+ and Na+, AMPA, but not NMDA, brought about changes in a guanine-nucleotide-binding protein (Gαi1) that inhibited pertussis toxin-mediated ADP-ribosylation of the protein in an in vitro assay. This effect was observed in intact neurons treated with AMPA as well as in isolated membranes exposed to AMPA, and was also found in MIN6 cells, which express functional AMPA receptors but have no metabotropic glutamate receptors. AMPA also inhibited forskolin-stimulated activity of adenylate cyclase in neurons, demonstrating that Gi proteins were activated. Moreover, both Gβγ blockage and co-precipitation experiments demonstrated that the modulation of the Gi protein arose from the association of Gαi1 with the glutamate receptor-1 (GluR1) subunit. These results suggest that, as well as acting as an ion channel, the AMPA receptor can exhibit metabotropic activity.

Development of immortalized human cerebromicrovascular endothelial cell line as an in vitro model of the human blood-brain barrier.

The objective of this study was to generate an immortal cell line representative of specialized human brain microvascular endothelia forming the blood–brain barrier (BBB) in vivo. Human capillary and microvascular endothelial cells (HCEC) were transfected with the plasmid pSV3‐neo coding for the SV40 large T antigen and the neomycin gene. The neomycin‐resistant transfected cells overcame proliferative senescence, and after a 6–8 wk period of crisis produced immortalization‐competent cell colonies. Single‐cell clones of near‐diploid genotype were isolated from these colonies, propagated, and characterized. Immortalized HCEC (SV‐HCEC) exhibited accelerated proliferation rates, but remained serum and anchorage dependent and retained the characteristic cobblestone morphology at confluence. SV‐HCEC displayed a stable nuclear expression of SV40 large T antigen, lacked the invasiveness of transformed cells, and maintained major phenotypic properties of early passage control cells including expression of factor VIII‐related antigen, uptake of acetylated low‐density lipoprotein, binding of fluorescently labeled lectins, expression of transferrin receptor and transferrin receptor‐mediated endocytosis, and high activities of the BBB‐specific enzymes alkaline phosphatase and γ‐glutamyl transpeptidase. The diffusion of radiolabeled sucrose across SV‐HCEC monolayers was fivefold lower than that observed with human lung microvascular endothelial cells. Furthermore, media conditioned by fetal human astrocytes increased the transendothelial electrical resistance of SV‐HCEC monolayers by 2.5‐fold. Therefore, this newly established human cell line expressing the specialized phenotype of BBB endothelium may serve as a readily available in vitro model for studying the properties of the human BBB.—Muruganandam, A., Herx, L. M., Monette, R., Durkin, J. P., Stanimirovic, D. B. Development of immortalized human cerebromicrovascular endothelial cell line as an in vitro model of the human blood–brain barrier. FASEB J. 1187–1197 (1997)

Functional Acetylcholine Muscarinic Receptor Subtypes in Human Brain Microcirculation: Identification and Cellular Localization

Acetylcholine is an important regulator of local cerebral blood flow. There is, however, limited information available on the possible sites of action of this neurotransmitter on brain intraparenchymal microvessels. In this study, a combination of molecular and functional approaches was used to identify which of the five muscarinic acetylcholine receptors (mAChR) are present in human brain microvessels and their intimately associated astroglial cells. Microvessel and capillary fractions isolated from human cerebral cortex were found by reverse transcriptase-polymerase chain reaction to express m2, m3, and, occasionally, m1 and m5 receptor subtypes. To localize these receptors to a specific cellular compartment of the vessel wall, cultures of human brain microvascular endothelial and smooth muscle cells were used, together with cultured human brain astrocytes. Endothelial cells invariably expressed m2 and m5 receptors, and occasionally the m1 receptor; smooth muscle cells exhibited messages for all except the m4 mAChR subtypes, whereas messages for all five muscarinic receptors were identified in astrocytes. In all three cell types studied, acetylcholine induced a pirenzepine-sensitive increase (62% to 176%, P < 0.05 to 0.01) in inositol trisphosphate, suggesting functional coupling of m1, m3, or m5 mAChR to a phospholipase C signaling cascade. Similarly, coupling of m2 or m4 mAChR to adenylate cyclase inhibition in endothelial cells and astrocytes, but not in smooth muscle cells, was demonstrated by the ability of carbachol to significantly reduce (44% to 50%, P < 0.05 to 0.01) the forskolin-stimulated increase in cAMP levels. This effect was reversed by the mAChR antagonist AF-DX 384. The results indicate that microvessels are able to respond to neurally released acetylcholine and that mAChR, distributed in different vascular and astroglial compartments, could regulate cortical perfusion and, possibly, blood–brain barrier permeability, functions that could become jeopardized in neurodegenerative disorders such as Alzheimers disease.

L-arginine induces dopamine release from the striatum in vivo

Recent reports indicate that induction of nitric oxide (NO) evokes dopamine (DA) release from the striatum in vitro. In this study, we used L-arginine (L-Arg) to demonstrate the in vivo stimulation of DA release from the striatum of Mongolian gerbils using microdialysis. The content of DA in the striatal extracellular fluid (ECF) increased 7–15-fold in the presence of L-Arg in the perfusate as compared with that of the controls (DA level in drug-free perfusate varied from 0.050 ± 0.009 to 0.092 ± 0.023 pmol 10 μUl−1). Simultaneous perfusion of L-Arg with nitro-L-arginine (NLA), an inhibitor of nitric oxide synthase, markedly reduced the L-Arg effect on DA release from the striatum. The NLA-perfused animals contained DA levels significantly lower than those observed in the control striatal dialysate. These findings indicate for the first time that DA release in vivo can be induced by L-Arg, the precursor of NO. The data strongly suggest that NO may modulate striatal DA release.

Method for isolation and molecular characterization of extracellular microvesicles released from brain endothelial cells

BackgroundIn addition to possessing intracellular vesicles, eukaryotic cells also produce extracellular microvesicles, ranging from 50 to 1000 nm in diameter that are released or shed into the microenvironment under physiological and pathological conditions. These membranous extracellular organelles include both exosomes (originating from internal vesicles of endosomes) and ectosomes (originating from direct budding/shedding of plasma membranes). Extracellular microvesicles contain cell-specific collections of proteins, glycoproteins, lipids, nucleic acids and other molecules. These vesicles play important roles in intercellular communication by acting as carrier for essential cell-specific information to target cells. Endothelial cells in the brain form the blood–brain barrier, a specialized interface between the blood and the brain that tightly controls traffic of nutrients and macromolecules between two compartments and interacts closely with other cells forming the neurovascular unit. Therefore, brain endothelial cell extracellular microvesicles could potentially play important roles in ‘externalizing’ brain-specific biomarkers into the blood stream during pathological conditions, in transcytosis of blood-borne molecules into the brain, and in cell-cell communication within the neurovascular unit.MethodsTo study cell-specific molecular make-up and functions of brain endothelial cell exosomes, methods for isolation of extracellular microvesicles using mass spectrometry-compatible protocols and the characterization of their signature profiles using mass spectrometry -based proteomics were developed.ResultsA total of 1179 proteins were identified in the isolated extracellular microvesicles from brain endothelial cells. The microvesicles were validated by identification of almost 60 known markers, including Alix, TSG101 and the tetraspanin proteins CD81 and CD9. The surface proteins on isolated microvesicles could potentially interact with both primary astrocytes and cortical neurons, as cell-cell communication vesicles. Finally, brain endothelial cell extracellular microvesicles were shown to contain several receptors previously shown to carry macromolecules across the blood brain barrier, including transferrin receptor, insulin receptor, LRPs, LDL and TMEM30A.ConclusionsThe methods described here permit identification of the molecular signatures for brain endothelial cell-specific extracellular microvesicles under various biological conditions. In addition to being a potential source of useful biomarkers, these vesicles contain potentially novel receptors known for delivering molecules across the blood–brain barrier.

Evidence that functional glutamate receptors are not expressed on rat or human cerebromicrovascular endothelial cells

Excitatory amino acids can modify the tone of cerebral vessels and permeability of the blood-brain barrier (BBB) by acting directly on endothelial cells of cerebral vessels or indirectly by activating receptors expressed on other brain cells. In this study we examined whether rat or human cerebromicrovascular endothelial cells (CEC) express ionotropic and metabotropic glutamate receptors. Glutamate and the glutamate receptor agonists N-methyl-d-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA), and kainate failed to increase [Ca2+]i in either rat or human microvascular and capillary CEC but elicited robust responses in primary rat cortical neurons, as measured by fura-2 fluorescence. The absence of NMDA and AMPA receptors in rat and human CEC was further confirmed by the lack of immunocytochemical staining of cells by antibodies specific for the AMPA receptor subunits GluR1, GluR2/3, and GluR4 and the NMDA receptor subunits NR1, NR2A, and NR2B. We failed to detect mRNA expression of the AMPA receptor subunits GluR1 to GluR4 or the NMDA receptor subunits NR11XX, NR10XX, and NR2A to NR2C in both freshly isolated rat and human microvessels and cultured CEC using reverse transcriptase polymerase chain reaction (RT-PCR). Cultured rat CEC expressed mRNA for KA1 or KA2 and GluR5 subunits. Primary rat cortical neurons were found to express GluR1 to GluR3 and NR1, NR2A, and NR2B by both immunocytochemistry and RT-PCR and KA1, KA2, GluR5, GluR6, and GluR7 by RT-PCR. Moreover, the metabotropic glutamate receptor agonist 1-amino-cyclopentyl-1S, 3R-dicorboxylate (1S,3R-trans-ACPD), while eliciting both inositol trisphosphate and [Ca2+]i increases and inhibiting forskolin-stimulated cyclic AMP in cortical neurons, was unable to induce either of these responses in rat or human CEC. These results strongly suggest that both rat and human CEC do not express functional glutamate receptors. Therefore, excitatory amino acid-induced changes in the cerebral microvascular tone and BBB permeability must be affected indirectly, most likely by mediators released from the adjacent glutamate-responsive cells.

Endothelin-1 receptor binding and cellular signal transduction in cultured human brain endothelial cells.

Abstract: The kinetic properties of endothelin‐1 (ET‐1) binding sites and the production of inositol phosphates (IPs; IP1, IP2, IP3), cyclic AMP, thromboxane B2, and prostaglandin F2α induced by various endothelins (ET‐1, ET‐2, ET‐3, and sarafotoxin S6b) were examined in endothelial cells derived from human brain microvessels (HBECs). The presence of both high‐ and low‐affinity binding sites for ET‐1 with KD1 = 122 pM and KD2 = 31 nM, and Bmax1 = 124 fmol/mg of protein and Bmax2 = 909 fmol/mg of protein, respectively, was demonstrated on intact HBECs. ET‐1 dose‐dependently stimulated IP accumulation with EC50 (IP3) = 0.79 nM, whereas ET‐3 was ineffective. The order of potency for displacing ET‐1 from high‐affinity binding sites (ET‐1 > ET‐2 > sarafotoxin S6b > ET‐3) correlated exponentially with the ability of respective ligands to induce IP3 formation. ET‐1‐induced IP3 formation by HBEC was inhibited by the ETA receptor antagonist, BQ123. The protein kinase C activator phorbol myristate ester dose‐dependently inhibited the ET‐1‐stimulated production of IPs, whereas pertussis toxin was ineffective. Cyclic AMP production by HBECs was enhanced by both phorbol myristate ester and ET‐1, and potentiated by combined treatment with ET‐1 and phorbol myristate ester. Data indicate that protein kinase C plays a role in regulating the ET‐1‐induced activation of phospholipase C, whereas interaction of different messenger systems may regulate ET‐1‐induced accumulation of cyclic AMP. ET‐1 also stimulated endothelial prostaglandin F2α production, suggesting that activation of phospholipase A2 is most likely secondary to IP3‐mediated intracellular calcium mobilization because both ET‐1‐induced IP3 and prostaglandin F2α were inhibited by BQ123. These findings are the first demonstration of ET‐1 (ETA‐type) receptors linked to phospholipase C and phospholipase A2 activation in HBECs.

Severe traumatic brain injury in children elevates glial fibrillary acidic protein in cerebrospinal fluid and serum

Objectives: 1) To determine the levels of glial fibrillary acidic protein (GFAP) in both cerebrospinal fluid and serum; 2) to determine whether serum GFAP levels correlate with functional outcome; and 3) to determine whether therapeutic hypothermia, as compared with normothermia, alters serum GFAP levels in children with severe traumatic brain injury (TBI). Design: Laboratory-based analyses; postrandomized, controlled trial. Setting: Four Canadian pediatric intensive care units and a university-affiliated laboratory. Patients: Twenty-seven children, aged 2–17 yrs, with severe TBI (Glasgow Coma Scale score of ≤8). Interventions: Hypothermia therapy (32.5°C) for 24 hrs with cooling started within 8 hrs of injury and rewarming at a rate of 0.5°C every 2 hrs or normothermia (37.0°C). Measurements and Main Results: GFAP was measured in cerebrospinal fluid and serum, using enzyme-linked immunosorbent assay. Levels of GFAP were maximal on day 1 post-TBI, with cerebrospinal fluid GFAP (15.5 ± 6.1 ng/mL) 25-fold higher than serum GFAP (0.6 ± 0.2 ng/mL). Cerebrospinal fluid GFAP normalized by day 7, whereas serum GFAP decreased gradually to reach a steady state by day 10. Serum GFAP measured on day 1 correlated with Pediatric Cerebral Performance Category scores determined at 6 months post-TBI (&rgr; = 0.527; p = .008) but failed to correlate with the injury scoring on admission, physiologic variables, or indices of injury measured on computerized tomography imaging. The areas under the receiver operating characteristic curves for pediatric intensive care unit day 1 serum GFAP in determining good outcome were 0.80 (pediatric cerebral performance category, 1–2; normal-mild disability) and 0.91 (pediatric cerebral performance category, 1–3; normal-moderate disability). For a serum GFAP cutoff level of 0.6 ng/mL, sensitivity and specificity were 88% to 90% and 43% to 71%, respectively. Serum GFAP levels were similar among children randomized to either therapeutic hypothermia or normothermia. Conclusions: GFAP was markedly elevated in cerebrospinal fluid and serum in children after severe TBI and serum GFAP measured on pediatric intensive care unit day 1 correlated with functional outcome at 6 months. Hypothermia therapy did not alter serum GFAP levels compared with normothermia after severe TBI in children. Serum GFAP concentration, together with other biomarkers, may have prognostic value after TBI in children.