The heparan sulfate proteoglycan agrin contributes to barrier properties of mouse brain endothelial cells by stabilizing adherens junctions

Barrier characteristics of brain endothelial cells forming the blood–brain barrier (BBB) are tightly regulated by cellular and acellular components of the neurovascular unit. During embryogenesis, the accumulation of the heparan sulfate proteoglycan agrin in the basement membranes ensheathing brain vessels correlates with BBB maturation. In contrast, loss of agrin deposition in the vasculature of brain tumors is accompanied by the loss of endothelial junctional proteins. We therefore wondered whether agrin had a direct effect on the barrier characteristics of brain endothelial cells. Agrin increased junctional localization of vascular endothelial (VE)-cadherin, β-catenin, and zonula occludens-1 (ZO-1) but not of claudin-5 and occludin in the brain endothelioma cell line bEnd5 without affecting the expression levels of these proteins. This was accompanied by an agrin-induced reduction of the paracellular permeability of bEnd5 monolayers. In vivo, the lack of agrin also led to reduced junctional localization of VE-cadherin in brain microvascular endothelial cells. Taken together, our data support the notion that agrin contributes to barrier characteristics of brain endothelium by stabilizing the adherens junction proteins VE-cadherin and β-catenin and the junctional protein ZO-1 to brain endothelial junctions.     

Immunohistochemical markers for quantitative studies of neurons and glia in human neocortex.

Reproducible visualization of neurons and glia in human brain is essential for quantitative studies of the cellular changes in neurological disease. However, immunohistochemistry in human brain specimens is often compromised because of prolonged fixation. To select cell lineage-specific antibodies for quantitative studies of neurons and the major types of glia, we used 29 different antibodies, different epitope retrieval methods, and different detection systems to stain tissue arrays of formalin-fixed human brain. The screening pointed at CD45/leukocyte common antigen (LCA), CD68(KP1), 2',3' cyclic nucleotide phosphatase (CNPase), glial fibrillary acidic protein (GFAP), HLA-DR, Ki67, neuronal nuclei (NeuN), p25alpha-antigen, and S100beta as candidates for future cell counting purposes, because these markers visualized specific neuronal and glial cell bodies. However, significant negative correlation between staining result and formalin fixation was observed by blinded scoring of staining for CD45/LCA, CNPase, GFAP, and NeuN in brain specimens fixed by immersion and stored up to 10 years in 4% formalin solution at room temperature, independent of donor sex and postmortem interval. In contrast, improved preservation of NeuN and CNPase staining, and full preservation of GFAP and CD45/LCA staining in tissue fixed by perfusion and stored for up to 3 years in 0.1% paraformaldehyde solution at 4C, indicated that immunohistochemistry can be performed in well-preserved biobank material.     

Intracellular domain of brain endothelial intercellular adhesion molecule-1 is essential for T lymphocyte-mediated signaling and migration

To examine the role of the ICAM-1 C-terminal domain in transendothelial T lymphocyte migration and ICAM-1-mediated signal transduction, mutant human (h)ICAM-1 molecules were expressed in rat brain microvascular endothelial cells. The expression of wild-type hICAM-1 resulted in a significant increase over basal levels in both adhesion and transendothelial migration of T lymphocytes. Endothelial cells (EC) expressing ICAM-1 in which the tyrosine residue at codon 512 was substituted with phenylalanine (hICAM-1(Y512F)) also exhibited increased lymphocyte migration, albeit less than that with wild-type hICAM-1. Conversely, the expression of truncated hICAM-1 proteins, in which either the intracellular domain was deleted (hICAM-1DeltaC) or both the intracellular and transmembrane domains were deleted through construction of a GPI anchor (GPI-hICAM-1), did not result in an increase in lymphocyte adhesion, and their ability to increase transendothelial migration was attenuated. Truncated hICAM-1 proteins were also unable to induce ICAM-1-mediated Rho GTPase activation. EC treated with cell-permeant penetratin-ICAM-1 peptides comprising human or rat ICAM-1 intracellular domain sequences inhibited transendothelial lymphocyte migration, but not adhesion. Peptides containing a phosphotyrosine residue were equipotent in inhibiting lymphocyte migration. These data demonstrate that the intracellular domain of ICAM-1 is essential for transendothelial migration of lymphocytes, and that peptidomimetics of the ICAM-1 intracellular domain can also inhibit this process. Such competitive inhibition of transendothelial lymphocyte migration in the absence of an affect on adhesion further implicates ICAM-1-mediated signaling events in the facilitation of T lymphocyte migration across brain EC. Thus, agents that mimic the ICAM-1 intracellular domain may be attractive targets for novel anti-inflammatory therapeutics.     

Simultaneous changes in cell quotas of microcystin, chlorophyll a, protein and carbohydrate during different growth phases of a batch culture experiment with Microcystis aeruginosa

The cell quotas of microcystin (Q_mcyst), protein (Q_prot), chlorophylla (Q_chloro) and carbohydrate (Q_carbo), as well as the net productionrates of these parameters, were determined during the exponentialand stationary phases in nine batch cultures of Microcystisaeruginosa (CYA 228) at light regimes from 33 to 53 µmolphotons m^–2 s^–1. The following results were obtained.(i) A parallel pattern was found in the changes of Q_mcyst, Q_prot,Q_chloro and Q_carbo during the entire growth cycle and significantcorrelations were recorded between Q_mcyst and Q_prot, Q_chloroand Q_carbo. (ii) The net microcystin production rate (µ_mcyst)was positively correlated with the specific cell division rate(µ_c), the chlorophyll production rate (µ_chloro)and the protein production rate. (iii) A significant inverselinear relationship was found between µ_c and Q_mcyst, i.e.cultures with a positive µ_c had a Q_mcyst between 110 and400 fg microcystin cell^–1, while declining cultures hadQ_mcyst values >400 fg microcystin cell^–1. Maximum variationin Q_mcyst within cultures was 3.5-fold. Collectively, the resultsshow that cells produced microcystin at rates approximatingthose needed to replace losses to daughter cells during divisionand that microcystin was produced in a similar way to proteinand chlorophyll, indicating a constitutive microcystin production.