Katerina Dorovini-Zis

Upregulation of intercellular adhesion molecule-1 (ICAM-1) expression in primary cultures of human brain microvessel endothelial cells by cytokines and lipopolysaccharide

The expression of intercellular adhesion molecule-1 (ICAM-1) by human cerebral endothelium was studied in primary cultures of human brain microvessel endothelial cells following treatment with bacterial lipopolysaccharide (LPS), tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta) and interferon-gamma (IFN-gamma). Surface expression of ICAM-1 was examined with the immunogold silver staining technique. Intact cerebral endothelial cells constitutively express low levels of ICAM-1. Stimulation with LPS and cytokines induces upregulation of ICAM-1 which is minimal with IFN-gamma and maximal with LPS or a combination of IFN-gamma and TNF-alpha. Upregulation of ICAM-1 expression is concentration- and time-dependent, is observed as early as 4 h following incubation and persists for up to 72 h in the continuous presence of LPS or cytokines. The ICAM-1 expression is not reversed by 3 days after removal of the LPS or cytokines. These findings may be relevant to the interactions between leukocytes and brain microvessel endothelial cells in inflammatory and demyelinating diseases of the CNS.

Notch Activation Results in Phenotypic and Functional Changes Consistent With Endothelial-to-Mesenchymal Transformation

Abstract— Various studies have identified a critical role for Notch signaling in cardiovascular development. In this and other systems, Notch receptors and ligands are expressed in regions that undergo epithelial-to-mesenchymal transformation. However, there is no direct evidence that Notch activation can induce mesenchymal transdifferentiation. In this study we show that Notch activation in endothelial cells results in morphological, phenotypic, and functional changes consistent with mesenchymal transformation. These changes include downregulation of endothelial markers (vascular endothelial [VE]-cadherin, Tie1, Tie2, platelet-endothelial cell adhesion molecule-1, and endothelial NO synthase), upregulation of mesenchymal markers (&agr;-smooth muscle actin, fibronectin, and platelet-derived growth factor receptors), and migration toward platelet-derived growth factor-BB. Notch-induced endothelial-to-mesenchymal transformation does not seem to require external regulation and is restricted to cells expressing activated Notch. Jagged1 stimulation of endothelial cells induces a similar mesenchymal transformation, and Jagged1, Notch1, and Notch4 are expressed in the ventricular outflow tract during stages of endocardial cushion formation. This is the first evidence that Jagged1-Notch interactions induce endothelial-to-mesenchymal transformation, and our findings suggest that Notch signaling may be required for proper endocardial cushion differentiation and/or vascular smooth muscle cell development.

Cytokines, nitric oxide, and cGMP modulate the permeability of an in vitro model of the human blood-brain barrier

The endothelial cells (EC) of the microvasculature in the brain form the anatomical basis of the blood-brain barrier (BBB). In the present study, the effects of agents that modify the permeability of a well-established in vitro model of the human BBB were studied. The monolayers formed by confluent human brain microvessel endothelial cell (HBMEC) cultures are impermeable to the macromolecule tracer horseradish peroxidase (HRP) and have high electrical resistance. Exposure of HBMEC to various cytokines including TNF-alpha, IL-1beta, interferon gamma (IFN-gamma), or lipopolysaccharide (LPS) decreased transendothelial electrical resistance (TEER) mainly by increasing the permeability of the tight junctions. Primary cultures of HBMEC express endothelial nitric oxide synthase (eNOS) and produce low levels of NO. Treatment with the NO donors sodium nitroprusside (SNP) and DETA NONOate or the cGMP agonist 8-Br-cGMP significantly increased monolayer resistance. Conversely, inhibition of soluble guanylyl cyclase with ODQ rapidly decreased the resistance, and pretreatment of HBMEC with Rp-8-CPT-cGMPS, an inhibitor of cGMP-dependent protein kinase, partially prevented the 8-Br-cGMP-induced increase in resistance. Furthermore, NO donors and 8-Br-cGMP could also reverse the increased permeability of the monolayers induced by IL-1beta, IFN-gamma, and LPS. These results indicate that NO can decrease the permeability of the human BBB through a mechanism at least partly dependent on cGMP production and cGMP-dependent protein kinase activation.

The neuropathology of fatal cerebral malaria in malawian children.

We examined the brains of 50 Malawian children who satisfied the clinical definition of cerebral malaria (CM) during life; 37 children had sequestration of infected red blood cells (iRBCs) and no other cause of death, and 13 had a nonmalarial cause of death with no cerebral sequestration. For comparison, 18 patients with coma and no parasitemia were included. We subdivided the 37 CM cases into two groups based on the cerebral microvasculature pathology: iRBC sequestration only (CM1) or sequestration with intravascular and perivascular pathology (CM2). We characterized and quantified the axonal and myelin damage, blood-brain barrier (BBB) disruption, and cellular immune responses and correlated these changes with iRBC sequestration and microvascular pathology. Axonal and myelin damage was associated with ring hemorrhages and vascular thrombosis in the cerebral and cerebellar white matter and brainstem of the CM2 cases. Diffuse axonal and myelin damage were present in CM1 and CM2 cases in areas of prominent iRBC sequestration. Disruption of the BBB was associated with ring hemorrhages and vascular thrombosis in CM2 cases and with sequestration in both CM1 and CM2 groups. Monocytes with phagocytosed hemozoin accumulated within microvessels containing iRBCs in CM2 cases but were not present in the adjacent neuropil. These findings are consistent with a link between iRBC sequestration and intravascular and perivascular pathology in fatal pediatric CM, resulting in myelin damage, axonal injury, and breakdown of the BBB.

In vitro adhesion and migration of T lymphocytes across monolayers of human brain microvessel endothelial cells: regulation by ICAM-1, VCAM-1, E-selectin and PECAM-1.

Increased lymphocyte traffic across an altered blood-brain barrier (BBB) is a prominent and early event in inflammatory and immune-mediated CNS diseases. The factors that control the entry of lymphocytes into the brain have not been fully elucidated. In this study, primary cultures of human brain microvessel endothelial cells (HBMEC) were used to investigate the role of endothelial cell (EC) adhesion molecules in the adhesion and migration of peripheral blood T lymphocytes across TNF-alpha treated and untreated monolayers. Adhesion of T cells to unstimulated HBMEC was minimal and few of the adherent cells migrated across the monolayers. Treatment of HBMEC with TNF-alpha augmented adhesion by 5-fold. The binding to activated EC was significantly, but not completely, inhibited by monoclonal antibodies (mAbs) to ICAM-1 and VCAM-1, whereas adhesion to unstimulated EC was blocked by mAb to ICAM-1 but not VCAM-1. Transendothelial migration of lymphocytes increased by up to 30-fold following treatment of HBMEC with TNF-alpha. Migration across activated monolayers, but not across untreated EC, was almost completely blocked by Ab to ICAM-1 and significantly inhibited by Abs to PECAM-1 and E-selectin. VCAM-1 was not utilized during transendothelial migration. Ultrastructurally, pseudopodia from lymphocytes contacted finger-like cytoplasmic projections on EC and eventually penetrated the EC cytoplasm at focal points along the apical surface. Migrating lymphocytes moved either through the EC cytoplasm or between adjacent EC across intercellular contacts. The overlying monolayers showed no evidence of disruption and intercellular junctions appeared intact over the migrated T cells. These studies indicate that adhesion and migration of T lymphocytes across the cerebral endothelial barrier are distinct processes that depend upon the activation state of EC and are controlled by diverse receptor-ligand interactions.

Regulation of CCL2 and CCL3 expression in human brain endothelial cells by cytokines and lipopolysaccharide

BackgroundChemokines are emerging as important mediators of CNS inflammation capable of activating leukocyte integrins and directing the migration of leukocyte subsets to sites of antigenic challenge. In this study we investigated the expression, release and binding of CCL2 (MCP-1) and CCL3 (MIP-1α) in an in vitro model of the human blood-brain barrier.MethodsThe kinetics of expression and cytokine upregulation and release of the β-chemokines CCL2 and CCL3 were studied by immunocytochemistry and enzyme-linked immunosorbent assay in primary cultures of human brain microvessel endothelial cells (HBMEC). In addition, the differential binding of these chemokines to the basal and apical endothelial cell surfaces was assessed by immunoelectron microscopy.ResultsUntreated HBMEC synthesize and release low levels of CCL2. CCL3 is minimally expressed, but not released by resting HBMEC. Treatment with TNF-α, IL-1β, LPS and a combination of TNF-α and IFN-γ, but not IFN-γ alone, significantly upregulated the expression and release of both chemokines in a time-dependent manner. The released CCL2 and CCL3 bound to the apical and basal endothelial surfaces, respectively. This distribution was reversed in cytokine-activated HBMEC resulting in a predominantly basal localization of CCL2 and apical distribution of CCL3.ConclusionsSince cerebral endothelial cells are the first resident CNS cells to contact circulating leukocytes, expression, release and presentation of CCL2 and CCL3 on cerebral endothelium suggests an important role for these chemokines in regulating the trafficking of inflammatory cells across the BBB in CNS inflammation.

Characterization of ABCB9, an ATP Binding Cassette Protein Associated with Lysosomes*

We have cloned full-length human and mouse cDNAs of ABCB9, which encodes a predicted multiple-spanning transmembrane domain and a nucleotide-binding domain with Walker motifs. It is therefore designated as a “half” ATP binding cassette (ABC) transporter. Northern analysis shows that the ABCB9 mRNA is expressed at a high level in testes and moderate levels in brain and spinal cord. A splice variant mRNA deleted in the last pair of predicted transmembrane segments was shown to be expressed in human tissues. Phylogenetic analysis indicates that ABCB9 is closely related to TAP1 and TAP2, two “half” ABC proteins found in endoplasmic reticulum. ABCB9 protein colocalized with the lysosomal markers, LAMP1 and LAMP2, in transfected cells. ABCB9 protein appears to be most highly expressed in the Sertoli cells of the seminiferous tubules in mouse and rat testes. These cells have high levels of phagocytosis and secretory activities. These findings pave the way for further investigation into the potential novel function of ABCB9 in lysosomes.

CD40 expressed by human brain endothelial cells regulates CD4+ T cell adhesion to endothelium

Recent evidence suggests that interactions between CD40 on antigen presenting cells (APC) and CD40L on T cells generate signals that result in the activation of APC. In this study, the expression and function of CD40 was investigated in primary cultures of human brain microvessel endothelial cells (HBMEC). Results revealed constitutive expression of CD40 on untreated HBMEC. Stimulation with TNF-alpha, IFN-gamma, LPS or combination of TNF-alpha and IFN-gamma significantly upregulated CD40. The majority of CD40 molecules were localized on the apical surface of EC. Incubation of HBMEC with soluble CD40L resulted in increased expression of the adhesion molecules E-selectin, VCAM-1 and ICAM-1. Consequently, the adhesion of both resting and anti-CD3 activated CD4+ T lymphocytes to CD40L treated HBMEC was significantly increased compared to unstimulated EC. The expression of CD40 by cerebral endothelium, and endothelial cell activation following binding of CD40 to its ligand, CD40L, suggest a potential mechanism by which activated CD40L expressing T cells could enhance adhesion and migration of inflammatory cells across the blood-brain barrier (BBB) to sites of inflammation in the human central nervous system (CNS).

Regulation by Cytokines and Lipopolysaccharide of E-selectin Expression by Human Brain Microvessel Endothelial Cells in Primary Culture

E-selectin is an adhesion molecule expressed on endothelial cells after treatment with inflammatory agents in vitro and in inflammatory diseases in vivo. Interactions between leukocytes and endothelial cells are mediated partly through this adhesion molecule. In this study, the kinetic expression of E-selectin by human cerebral endothelium was studied under standard conditions and following treatment of primary cultures with bacterial lipopolysaccharide (LPS), tumor necrosis factora (TNF-a), interleukin-1 (3 (IL-1(

Adhesion and migration of polymorphonuclear leukocytes across human brain microvessel endothelial cells are differentially regulated by endothelial cell adhesion molecules and modulate monolayer permeability

), and interferon-7 (IFN-7). Surface expression of E-selectin was detected by immunocytochemistry, ELISA and immunoelectron microscopy. Untreated human cerebral endothelial cells constitutively expressed low levels of E-selectin. Treatment with LPS, TNF-a and IL-ip increased the mean level of E-selectin expression per cell and the percentage of cells expressing E-selectin, in a time- and concentration-dependent manner. E-selectin expression was maximal by 4 h post-stimulation and returned to unstimulated levels by 48 h. LPS and TNF-a were most effective followed by IL-1(3, while the IFN-7 had no effect on E-selectin expression. Immunoelectron microscopy demonstrated that E-selectin was preferentially expressed on the apical surface of unstimulated and TNF-a treated cells. Cytokine stimulation induced a several-fold increase of E-selectin expression on the apical and to a lesser extent on the basal cell surface. Modulation of Eselectin expression on cerebral endothelium by inflammatory cytokines suggests a potentially important role of this adhesion molecule in the recruitment of leukocytes in central nervous system (CNS) inflammation.