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ABSTRACT: Junctional adhesion molecule A (JAM-A) is a unique tight junction (TJ) transmembrane protein that under basal conditions maintains endothelial cell-cell interactions but under inflammatory conditions acts as a leukocyte adhesion molecule. This study investigates the fate of JAM-A during inflammatory TJ complex remodeling and paracellular route formation in brain endothelial cells. The chemokine (C-C motif) ligand 2 (CCL2) induced JAM-A redistribution from the interendothelial cell area to the apical surface, where JAM-A played a role as a leukocyte adhesion molecule participating in transendothelial cell migration of neutrophils and monocytes. JAM-A redistribution was associated with internalization via macropinocytosis during paracellular route opening. A tracer study with dextran-Texas Red indicated that internalization occurred within a short time period (~10 min) by dextran-positive vesicles and then became sorted to dextran-positive/Rab34-positive/Rab5-positive vesicles and then Rab4-positive endosomes. By ~20 min, most internalized JAM-A moved to the brain endothelial cell apical membrane. Treatment with a macropinocytosis inhibitor, 5-(N-ethyl-N-isopropyl)amiloride, or Rab5/Rab4 depletion with small interfering RNA oligonucleotides prevented JAM-A relocalization, suggesting that macropinocytosis and recycling to the membrane surface occur during JAM-A redistribution. Analysis of the signaling pathways indicated involvement of RhoA and Rho kinase in JAM-A relocalization. These data provide new insights into the molecular and cellular mechanisms involved in blood-brain barrier remodeling during inflammation.
Molecular and cellular biology 06/2012; 32(17):3414-27. · 6.06 Impact Factor
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09/2011; , ISBN: 978-953-307-680-5
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ABSTRACT: Claudin-5 is a transmembrane tight junction protein highly expressed in brain endothelial cells, the site of the blood-brain barrier. The properties of the brain endothelial tight junction complex are considered to be dependent on claudin-5 cell-cell interaction, putting this protein in a position to play a major role in the maintenance of brain endothelial barrier integrity. Thus, alterations in claudin-5 function can lead to "opening" of the paracellular route and increased brain endothelial barrier permeability. Recent work from the authors's laboratory has established that caveolae-dependent internalization/recycling of claudin-5 is a mechanism underlying transient increases in brain endothelial paracellular permeability in the presence of pro-inflammatory mediators. The biochemical and microscopic techniques presented here were used to investigate trafficking of claudin-5 during those changes in paracellular permeability.
Methods in molecular biology (Clifton, N.J.) 01/2011; 762:303-20.
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ABSTRACT: A substantial body of evidence suggests that nicotine adversely affects cerebral blood flow and the blood-brain barrier and is a risk factor for stroke. The present study investigated the effect of nicotine on cerebrovascular endothelium under basal and ischemia/reperfusion injury under in vivo condition. Nicotine (2 mg/kg sc) was administered to mice over 14 days, which resulted in plasma nicotine levels of ∼100 ng/ml, reflecting plasma concentrations in average to heavy smokers. An analysis of the phenotype of isolated brain microvessels after nicotine exposure indicated higher expression of inflammatory mediators, cytokines (IL-1β, TNF-α, and IL-18), chemokines (CCL2 and CX(3)CL1), and adhesion molecules (ICAM-1, VCAM-1, and P-selectins), and this was accompanied by enhanced leukocyte infiltration into brain during ischemia/reperfusion (P < 0.01). Nicotine had a profound effect on ischemia/reperfusion injury; i.e., increased brain infarct size (P < 0.01), worse neurological deficits, and a higher mortality rate. These experiments illuminate, for the first time, how nicotine regulates brain endothelial cell phenotype and postischemic inflammatory response at the brain-vascular interface.
AJP Heart and Circulatory Physiology 01/2011; 300(4):H1518-29. · 3.71 Impact Factor
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ABSTRACT: Disturbance of the tight junction (TJ) complexes between brain endothelial cells leads to increased paracellular permeability, allowing leukocyte entry into inflamed brain tissue and also contributing to edema formation. The current study dissects the mechanisms by which a chemokine, CCL2, induces TJ disassembly. It investigates the potential role of selective internalization of TJ transmembrane proteins (occludin and claudin-5) in increased permeability of the brain endothelial barrier in vitro. To map the internalization and intracellular fate of occludin and claudin-5, green fluorescent protein fusion proteins of these TJ proteins were generated and imaged by fluorescent microscopy with simultaneous measurement of transendothelial electrical resistance. During CCL2-induced reductions in transendothelial electrical resistance, claudin-5 and occludin became internalized via caveolae and further processed to early (EEA1+) and recycling (Rab4+) endosomes but not to late endosomes. Western blot analysis of fractions collected from a sucrose gradient showed the presence of claudin-5 and occludin in the same fractions that contained caveolin-1. For the first time, these results suggest an underlying molecular mechanism by which the pro-inflammatory chemokine CCL2 mediates brain endothelial barrier disruption during CNS inflammation.
Journal of Biological Chemistry 06/2009; 284(28):19053-66. · 4.77 Impact Factor
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ABSTRACT: Brain edema (BE) is an uncommon but life-threatening complication of severe diabetic ketoacidosis (DKA) and its treatment. Despite advances in treatment of DKA, the pathogenesis of both initiation and progression of the associated BE is unclear. In the present study we examined the blood brain barrier (BBB) integrity and the potential involvement of the inflammatory mediators in BBB breakdown in two cases of fatal BE associated with DKA. In both cases there were typical signs of disruption of the BBB manifested by the absence of tight junction proteins (occludin, claudin-5, ZO-1 and JAM-1) in the parenchymal blood vessels, as well as albumin extravasation in examined brain areas. The neuroinflammatory markers chemokine CCL2, NF-kappaB and nitrotyrosine were localized in the perivascular areas of the disrupted BBB and diffusely distributed in the brain parenchyma. Our data indicate that neuroinflammation plays a role in the BBB disruption of the fatal BE of DKA.
Brain research 01/2009; 1254:138-48. · 2.46 Impact Factor
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ABSTRACT: The blood-brain barrier (BBB) is a highly specialized structural and biochemical barrier that regulates the entry of blood-borne molecules into brain, and preserves ionic homeostasis within the brain microenvironment. BBB properties are primarily determined by junctional complexes between the cerebral endothelial cells. These complexes are comprised of tight and adherens junctions. Such restrictive angioarchitecture at the BBB reduces paracellular diffusion, while minimal vesicle transport activity in brain endothelial cells limits transcellular transport. Under normal conditions, this largely prevents the extravasation of large and small solutes (unless specific transporters are present) and prevents migration of any type of blood-borne cell. However, this is changed in many pathological conditions. There, BBB disruption ("opening") can lead to increased paracellular permeability, allowing entry of leukocytes into brain tissue, but also contributing to edema formation. In parallel, there are changes in the endothelial pinocytotic vesicular system resulting in the uptake and transfer of fluid and macromolecules into brain parenchyma. This review highlights the route and possible factors involved in BBB disruption in a variety of neuropathological disorders (e.g. CNS inflammation, Alzheimer's disease, Parkinson's disease, epilepsy). It also summarizes proposed signal transduction pathways that may be involved in BBB "opening".
Current Neuropharmacology 10/2008; 6(3):179-92. · 2.85 Impact Factor
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ABSTRACT: The chemokine, monocyte chemoattractant protein-1 (CCL2), is a major factor driving leukocyte infiltration into the brain parenchyma in a variety of neuropathologic conditions associated with inflammation, including stroke. In addition, recent studies indicate that CCL2 and its receptor (CCR2) could have an important role in regulating blood-brain barrier (BBB) permeability. This study evaluated the role of the CCL2/CCR2 axis in regulating postischemic inflammation, BBB breakdown, and vasogenic edema formation.
CCR2(-/-) and CCR2(+/+) mice were subjected to focal transient cerebral ischemia. BBB permeability and brain edema formation were observed at days 1 and 5 of reperfusion by evaluating the product surface area for fluorescein isothiocyanate-albumin and measuring water and electrolyte contents. Immunohistochemistry was used to assess leukocyte infiltration. cDNA gene and protein arrays for inflammatory cytokines were used to assess inflammatory profiles in CCR2(+/+) and CCR2(-/-) mice.
CCR2(-/-) mice had reduced infarct sizes and significantly reduced BBB permeability and brain edema formation in the affected ischemic hemisphere compared with CCR2(+/+) mice. This reduction in injury was closely associated with reduced infiltration of not only monocytes but also neutrophils (7- and 4-fold decreases, respectively). In addition, CCR2(-/-) mice had reduced expression/production of inflammatory cytokines during reperfusion.
These data suggest that inhibiting the CCL2/CCR2 axis affects brain reperfusion outcome by reducing brain edema, leukocyte infiltration, and inflammatory mediator expression.
Stroke 05/2007; 38(4):1345-53. · 5.73 Impact Factor
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ABSTRACT: Although recent studies have suggested that CC chemokine CCL2 may directly affect the angiogenesis, the signaling events involved in such regulation remain to be determined. This study investigated a potential signal mechanism involved in CCL2-induced angiogenesis. Our in vitro and in vivo (hemangioma model of angiogenesis) experiments confirmed earlier findings that CCL2 can induce angiogenesis directly. Using a gene array analysis, CCL2 was found to induce expression of several angiogenic factors in brain endothelial cells. Among the most prominent was an up-regulation in Ets-1 transcription factor. CCL2 induced a significant increase in Ets-1 mRNA and protein expression as well as Ets-1 DNA-binding activity. Importantly, Ets-1 antisense oligonucleotide markedly abrogated in vitro CCL2-induced angiogenesis, suggesting that Ets-1 is critically involved in this process. Activation of Ets-1 by CCL2 further regulated some of Ets-1 target molecules including beta(3) integrins. CCL2 induced significant up-regulation of beta(3) mRNA and protein expression, and this effect of CCL2 was prevented by the Ets-1 antisense oligonucleotide. The functional regulation of Ets-1 activity by CCL2 was dependent on ERK-1/2 cascade. Inhibition of ERK1/2 activity by PD98509 prevented CCL2-induced increases in Ets-1 DNA-binding activity and Ets-1 mRNA expression. Based on these findings, we suggest that Ets-1 transcription factor plays a critical role in CCL2 actions on brain endothelial cells and CCL2-induced angiogenesis.
The Journal of Immunology 09/2006; 177(4):2651-61. · 5.79 Impact Factor
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ABSTRACT: The chemokine CCL2 is considered as one of the main effectors driving postischemic infiltration of monocytes into the brain parenchyma. New experimental data, however, suggest that CCL2 could also participate in blood-brain barrier (BBB) 'opening' during the transmigration of monocytes. The current study examines the role of CCL2 in regulating BBB permeability after ischemia in vitro. To address this issue, an in vitro BBB model (coculture of astrocytes and brain endothelial cells) was subjected to 5 h of oxygen glucose deprivation, followed by reoxgenation (in vitro ischemia/reperfusion (I/R)) for 0 to 48 h. During reperfusion, there was a biphasic enhancement of barrier permeability, with a 200-fold increase in barrier permeability to FITC-albumin at 6 h and a further period of disruption around 24 h. The latter coincided with increased secretion of CCL2 by both astrocytes and brain endothelial cells and increased levels of the CCL2 receptor, CCR2. Applying antisense oligonucleotide or neutralizing antibody to block CCL2 significantly decreased I/R-induced enhancement of BBB permeability (approximately twofold) and redistribution of tight-junction (TJ) proteins (occludin, zonula occluden-1, 2, claudin-5). Similarly, absence of CCR2 from endothelial cells caused stabilization of TJ complexes and decreased the permeability of brain endothelial barrier during in vitro I/R. These data suggest CCL2/CCR2 has an important role in regulating brain endothelial permeability and might be a potential novel therapeutic target for stroke.
Journal of Cerebral Blood Flow & Metabolism 07/2006; 26(6):797-810. · 5.01 Impact Factor
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ABSTRACT: Monocyte chemoattractant protein-1 (MCP-1 or CCL2) regulates blood-brain barrier permeability by inducing morphological and biochemical alterations in the tight junction (TJ) complex between brain endothelial cells. The present study used cultured brain endothelial cells to examine the signaling networks involved in the redistribution of TJ proteins (occludin, ZO-1, ZO-2, claudin-5) by CCL2. The CCL2-induced alterations in the brain endothelial barrier were associated with de novo Ser/Thr phosphorylation of occludin, ZO-1, ZO-2, and claudin-5. The phosphorylated TJ proteins were redistributed/localized in Triton X-100-soluble as well as Triton X-100-insoluble cell fractions. Two protein kinase C (PKC) isoforms, PKCalpha and PKCzeta, had a significant impact on this event. Inhibition of their activity using dominant negative mutants PKCalpha-DN and PKCzeta-DN diminished CCL2 effects on brain endothelial permeability. Previous data indicate that Rho/Rho kinase signaling is involved in CCL2 regulation of brain endothelial permeability. The interactions between the PKC and Rho/Rho kinase pathways were therefore examined. Rho, PKCalpha, and PKCzeta activities were knocked down using dominant negative mutants (T17Rho, PKCalpha-DN, and PKCzeta-DN, respectively). PKCalpha and Rho, but not PKCzeta and Rho, interacted at the level of Rho, with PKCalpha being a downstream target for Rho. Double transfection experiments using dominant negative mutants confirmed that this interaction is critical for CCL2-induced redistribution of TJ proteins. Collectively these data suggest for the first time that CCL2 induces brain endothelial hyperpermeability via Rho/PKCalpha signal pathway interactions.
Journal of Biological Chemistry 04/2006; 281(13):8379-88. · 4.77 Impact Factor
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ABSTRACT: The present study was designed to elucidate the effects of the chemokine monocyte chemoattractant protein (MCP-1) on blood-brain barrier (BBB) permeability. Experiments were conducted under in vitro conditions (coculture of brain endothelial cells and astrocytes) to study the cellular effects of MCP-1 and under in vivo conditions (intracerebral and intracerebroventricular administration of MCP-1) to study the potential contribution of MCP-1 to BBB disruption in vivo. Our results showed that MCP-1 induces a significant increase in the BBB permeability surface area product for fluorescein isothiocyanate (FITC)-albumin under in vivo conditions, particularly during prolonged (3 or 7 days) exposure (0.096+/-0.008 versus 0.031+/-0.005 microL/g min in controls at 3 days, P<0.001). Monocyte chemoattractant protein-1 also enhanced (17-fold compared with control) the permeability of the in vitro BBB (coculture) model. At the cellular level, MCP-1 causes alteration of tight junction (TJ) proteins in endothelial cells (redistribution of TJ proteins determined by Western blotting and loss of immunostaining for occludin, claudin-5, ZO-1, ZO-2). Monocyte chemoattractant protein-1-induced alterations in BBB permeability are mostly realized through the CCR2 receptor. Absence of CCR2 diminishes any effect of MCP-1 on BBB permeability in vitro and in vivo. The permeability surface area product for FITC-albumin after 3 days exposure to MCP-1 was 0.096+/-0.006 and 0.032+/-0.007 microL/g min, in CCR2+/+ and CCR2-/- mice, respectively (P<0.001). Monocytes/macrophages also participate in MCP-1-induced alterations in BBB permeability in vivo. Monocytes/macrophages depletion (by clodronate liposomes) reduced the effect of MCP-1 on BBB permeability in vivo approximately 2 fold. Our results suggest that, besides its main function of recruiting leukocytes at sites of inflammation, MCP-1 also plays a role in 'opening' the BBB.
Journal of Cerebral Blood Flow & Metabolism 06/2005; 25(5):593-606. · 5.01 Impact Factor
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ABSTRACT: 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).
Journal of Cerebral Blood Flow & Metabolism 12/2003; 23(11):1348-55. · 5.01 Impact Factor
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ABSTRACT: The expression of the monocyte chemoattractant protein-1 (MCP-1) receptor CCR2 by brain endothelial cells suggests that MCP-1 may have other functions than purely driving leukocyte migration into brain parenchyma during inflammation. This study examines one of these potential novel roles of MCP-1 regulation of endothelial permeability using primary cultures of mouse brain endothelial cells. MCP-1 induces reorganization of actin cytoskeleton (stress fiber formation) and redistribution of tight junction proteins, ZO-1, ZO-2 occludin and claudin-5, from the Triton X-100-soluble to the Triton X-100-insoluble fractions. These morphological changes are associated with a decrease in transendothelial electrical membrane resistance and an increase in [14C]inulin permeability. MCP-1 did not induce these events in brain endothelial cells prepared from mice genotype CCR2-/-. The Rho kinase inhibitor Y27632 and inhibition of Rho (C3 exoenzyme, and dominant negative mutant of Rho, RhoT19N) prevented MCP-1-induced stress fiber assembly, reorganization of tight junction proteins and alterations in endothelial permeability. In all, this suggests that a small GTPase Rho and Rho kinase have a pivotal role in MCP-1-induced junction disarrangement. These data are the first to strongly suggest that MCP-1, via CCR2 present on brain endothelial cells, contributes to increased brain endothelial permeability.
Journal of Cell Science 12/2003; 116(Pt 22):4615-28. · 6.11 Impact Factor
