Evaluation of soluble junctional adhesion molecule-A as a biomarker of human brain endothelial barrier breakdown

Background

An inducible release of soluble junctional adhesion molecule-A (sJAM-A) under pro-inflammatory conditions was described in cultured non-CNS endothelial cells (EC) and increased sJAM-A serum levels were found to indicate inflammation in non-CNS vascular beds. Here we studied the regulation of JAM-A expression in cultured brain EC and evaluated sJAM-A as a serum biomarker of blood-brain barrier (BBB) function.

Methodology/Principal Findings

As previously reported in non-CNS EC types, pro-inflammatory stimulation of primary or immortalized (hCMEC/D3) human brain microvascular EC (HBMEC) induced a redistribution of cell-bound JAM-A on the cell surface away from tight junctions, along with a dissociation from the cytoskeleton. This was paralleled by reduced immunocytochemical staining of occludin and zonula occludens-1 as well as by increased paracellular permeability for dextran 3000. Both a self-developed ELISA test and Western blot analysis detected a constitutive sJAM-A release by HBMEC into culture supernatants, which importantly was unaffected by pro-inflammatory or hypoxia/reoxygenation challenge. Accordingly, serum levels of sJAM-A were unaltered in 14 patients with clinically active multiple sclerosis compared to 45 stable patients and remained unchanged in 13 patients with acute ischemic non-small vessel stroke over time.

Conclusion

Soluble JAM-A was not suited as a biomarker of BBB breakdown in our hands. The unexpected non-inducibility of sJAM-A release at the human BBB might contribute to a particular resistance of brain EC to inflammatory stimuli, protecting the CNS compartment.     

Uncertainty and spatial variability in characterization factors for aquatic acidification at the global scale

Purpose

Characterization factors (CFs) quantifying the potential impact of acidifying emissions on inland aquatic environments in life cycle assessment are typically available on a generic level. The lack of spatial differentiation may weaken the relevance of generic CFs since it was shown that regional impact categories such as aquatic acidification were influenced by the surroundings of the emission location. This paper presents a novel approach for the development of spatially differentiated CFs at a global scale for the aquatic acidification impact category.

Methods

CFs were defined as the change in relative decrease of lake fish species richness due to a change in acidifying chemicals emissions. The characterization model includes the modelling steps linking emission to atmospheric acid deposition (atmospheric fate factor) change, which lead to lake H⁺ concentration (receiving environment fate factor) change and a decrease in relative fish species richness (effect factor). We also evaluated the significance of each factor (i.e. atmospheric fate, receiving environment fate and effects) to the overall CFs spatial variability and parameter uncertainty.

Results and discussion

The highest CFs were found for emissions occurring in Canada, Scandinavia and the northern central Asia because of the extensive lake areas in these regions (lake areas being one of the parameters of the CFs; the bigger the lake areas, the higher the CFs). The CFs’ spatial variability ranged over 5, 6 and 8 orders of magnitude for NO_x, SO₂ and NH₃ emissions, respectively. We found that the aquatic receiving environment fate factor is the dominant contributor to the overall spatial variability of the CFs, while the effect factors contributed to 98 % of the total parameter uncertainty.

Conclusions

The resulting characterization model and factors enable a consistent evaluation of spatially explicit acidifying emissions impacts at the global scale.     

A G protein–coupled receptor and the intracellular synthase of its agonist functionally cooperate

Export of newly synthesized G protein–coupled receptors (GPCRs) remains poorly characterized. We show in this paper that lipocalin-type prostaglandin D₂ (PGD₂) synthase (L-PGDS) interacts intracellularly with the GPCR DP1 in an agonist-independent manner. L-PGDS promotes cell surface expression of DP1, but not of other GPCRs, in HEK293 and HeLa cells, independent of L-PGDS enzyme activity. In addition, formation of a DP1–Hsp90 complex necessary for DP1 export to the cell surface is dependent on the interaction between L-PGDS and the C-terminal MEEVD residues of Hsp90. Surprisingly, PGD₂ synthesis by L-PGDS is promoted by coexpression of DP1, suggesting a possible intracrine/autocrine signaling mechanism. In this regard, L-PGDS increases the formation of a DP1–ERK1/2 complex and increases DP1-mediated ERK1/2 signaling. Our findings define a novel cooperative mechanism in which a GPCR (DP1) promotes the activity of the enzyme (L-PGDS) that produces its agonist (PGD₂) and in which this enzyme in turn acts as a cofactor (of Hsp90) to promote export and agonist-dependent activity of the receptor.     

Overexpression of CD44 in Neural Precursor Cells Improves Trans- Endothelial Migration and Facilitates Their Invasion of Perivascular Tissues In Vivo

Neural precursor (NPC) based therapies are used to restore neurons or oligodendrocytes and/or provide neuroprotection in a large variety of neurological diseases. In multiple sclerosis models, intravenously (i.v) -delivered NPCs reduced clinical signs via immunomodulation. We demonstrated recently that NPCs were able to cross cerebral endothelial cells in vitro and that the multifunctional signalling molecule, CD44 involved in trans-endothelial migration of lymphocytes to sites of inflammation, plays a crucial role in extravasation of syngeneic NPCs. In view of the role of CD44 in NPCs trans-endothelial migration in vitro, we questioned presently the benefit of CD44 overexpression by NPCs in vitro and in vivo, in EAE mice. We show that overexpression of CD44 by NPCs enhanced over 2 folds their trans-endothelial migration in vitro, without impinging on the proliferation or differentiation potential of the transduced cells. Moreover, CD44 overexpression by NPCs improved significantly their elongation, spreading and number of filopodia over the extracellular matrix protein laminin in vitro. We then tested the effect of CD44 overexpression after i.v. delivery in the tail vein of EAE mice. CD44 overexpression was functional in vivo as it accelerated trans-endothelial migration and facilitated invasion of HA expressing perivascular sites. These in vitro and in vivo data suggest that CD44 may be crucial not only for NPC crossing the endothelial layer but also for facilitating invasion of extravascular tissues.     

S-adenosylhomocysteine Hydrolase Participates in DNA Methylation Inheritance

DNA (cytosine-5) methyltransferase 1 (DNMT1) is essential for mammalian development and maintenance of DNA methylation following DNA replication in cells. The DNA methylation process generates S-adenosyl-l-homocysteine, a strong inhibitor of DNMT1. Here we report that S-adenosylhomocysteine hydrolase (SAHH/AHCY), the only mammalian enzyme capable of hydrolyzing S-adenosyl-l-homocysteine binds to DNMT1 during DNA replication. SAHH enhances DNMT1 activity in vitro, and its overexpression in mammalian cells led to hypermethylation of the genome, whereas its inhibition by adenosine periodate or siRNA-mediated knockdown resulted in hypomethylation of the genome. Hypermethylation was consistent in both gene bodies and repetitive DNA elements leading to aberrant gene regulation. Cells overexpressing SAHH specifically up-regulated metabolic pathway genes and down-regulated PPAR and MAPK signaling pathways genes. Therefore, we suggest that alteration of SAHH level affects global DNA methylation levels and gene expression.     

Coupling of ETB Endothelin Receptor to Mitogen-Activated Protein Kinase Stimulation and DNA Synthesis in Primary Cultures of Rat Astrocytes

Abstract: Astrocytes have been shown to express endothelin (ET) receptors functionally coupled, via different heterotrimeric G proteins, to several intracellular pathways. To assess the relative contribution of each subtype in the astrocytic responses to ET-1, effects of BQ123, an antagonist selective for the ET receptor subtype A (ET_A-R), and IRL1620, an agonist selective for the ET receptor subtype B (ET_B-R), were investigated in primary cultures of rat astrocytes. Binding experiments indicated that the ET_B-R is the predominant subtype in these cells. Inhibition of forskolin-stimulated cyclic AMP production was observed under ET_B-R stimulation. Bordetella pertussis toxin (PTX) pretreatment completely abolished this effect, indicating that this pathway is coupled to the ET_B-R via G_i protein. Increases of tyrosine phosphorylation of cellular proteins, stimulation of mitogen-activated protein kinase (MAPK), and DNA synthesis were also found to be mediated by the ET_B-R, but through PTX-insensitive G protein. IRL1620-induced MAPK activation involved the adapter proteins Shc and Grb2 and the serine/threonine kinase Raf-1. This study reveals that the various effects of ET-1 in astrocytes are mediated by the ET_B-R, which couples to multiple signaling pathways including the MAPK cascade.     

Axonal Transport of the Molecular Forms of Acetylcholinesterase in Chick Sciatic Nerve

Acetylcholinesterase (AChE) polymorphism was studied in the sciatic nerve of 4-week-old Leghorn chicks, by sucrose gradient sedimentation analysis. Four main AChE molecular forms were found with sedimentation coefficients of 5S, 7.5S, 11.5S and 20S respectively. Axonal transport of each of these forms was investigated on the basis of the enzyme accumulation kinetics measured on both sides of nerve transections and of the enzyme redistribution kinetics in nerve segments isolated in vivo. After nerve transection, 11.5S and 20S forms accumulated faster in the anterograde than in the retrograde direction and also much faster than 5S and 7.5S forms in the anterograde direction. Retrograde accumulations of 5S and 7.5S were faint or negligible. In addition, 1 h after nerve cutting, the accumulation rates for 11.5S and 20S forms (but not for 5S and 7.5S) fell, in both directions, to about one-third of their initial values, probably owing to reversal of axonal transport at the axotomy site. Local protein synthesis inhibition by cycloheximide did not affect the accumulation of 11.5S and 20S in front of a transection, at least during the first hours, but reduced that of 5S and 7.5S by about 40%. In isolated nerve segments in vivo, the rapidly mobile fraction of AChE was estimated to constitute 23% of the total enzyme activity present in the nerve, 14% of it moving in an anterograde and 9% in a retrograde direction. A small amount of 11.5S molecules (approx. 20%) was in rapid transit (two-thirds in the anterograde and one-third in the retrograde direction), whereas almost all the 20S--about 90%--migrated rapidly (two-thirds forwards and one-third backwards). Anterograde velocities of 408 +/- 94 and 411 +/- 161 mm/day respectively were estimated for the 11.5S and 20S forms. Their respective retrograde velocities were 175 +/- 85 and 145 +/- 107 mm/day. Assuming that the totality of 5S and 7.5S molecules are moving in the anterograde direction, their accumulation rates were consistent with the average anterograde velocities of 2.9 +/- 1.3 and 5.1 +/- 1.4 mm/day, respectively.