Transcriptional Profiling of Human Brain Endothelial Cells Reveals Key Properties Crucial for Predictive In Vitro Blood-Brain Barrier Models

Brain microvascular endothelial cells (BEC) constitute the blood-brain barrier (BBB) which forms a dynamic interface between the blood and the central nervous system (CNS). This highly specialized interface restricts paracellular diffusion of fluids and solutes including chemicals, toxins and drugs from entering the brain. In this study we compared the transcriptome profiles of the human immortalized brain endothelial cell line hCMEC/D3 and human primary BEC. We identified transcriptional differences in immune response genes which are directly related to the immortalization procedure of the hCMEC/D3 cells. Interestingly, astrocytic co-culturing reduced cell adhesion and migration molecules in both BECs, which possibly could be related to regulation of immune surveillance of the CNS controlled by astrocytic cells within the neurovascular unit. By matching the transcriptome data from these two cell lines with published transcriptional data from freshly isolated mouse BECs, we discovered striking differences that could explain some of the limitations of using cultured BECs to study BBB properties. Key protein classes such as tight junction proteins, transporters and cell surface receptors show differing expression profiles. For example, the claudin-5, occludin and JAM2 expression is dramatically reduced in the two human BEC lines, which likely explains their low transcellular electric resistance and paracellular leakiness. In addition, the human BEC lines express low levels of unique brain endothelial transporters such as Glut1 and Pgp. Cell surface receptors such as LRP1, RAGE and the insulin receptor that are involved in receptor-mediated transport are also expressed at very low levels. Taken together, these data illustrate that BECs lose their unique protein expression pattern outside of their native environment and display a more generic endothelial cell phenotype. A collection of key genes that seems to be highly regulated by the local surroundings of BEC within the neurovascular unit are presented and discussed

Effects of a Fully Humanized Type II Anti-CD20 Monoclonal Antibody on Peripheral and CNS B Cells in a Transgenic Mouse Model of Multiple Sclerosis

Successful therapy with anti-CD20 monoclonal antibodies (mAbs) has reinforced the key role of B cells in the immunopathology of multiple sclerosis (MS). This study aimed to determine the effects of a novel class of anti-CD20 mAbs on vascular and extravascular central nervous system (CNS)-infiltrating B cells in experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Male hCD20xhIgR3 mice and wild-type C57BL/6 (B6) mice were immunized with human myelin oligodendrocyte glycoprotein (MOG)1–125 to induce EAE. While hCD20xhIgR3 mice were injected intravenously with an anti-human CD20 mAb (5 mg/kg) (rituximab (a type I anti-CD20 mAb) or obinutuzumab (a type II anti-CD20 mAb), B6 mice received the anti-mouse CD20 antibody 18B12. Neither mAb affected clinical disease or serum antibody levels. Obinutuzumab and rituximab had an impact on splenic and CNS-infiltrated B cells with slightly differential depletion efficacy. Additionally, obinutuzumab had beneficial effects on spinal cord myelination. B cell depletion rates in the 18B12/B6 model were comparable with those observed in obinutuzumab-treated hCD20xhIgR3 mice. Our results demonstrate the usefulness of anti-CD20 mAbs for the modulation of B cell-driven peripheral immune response and CNS pathology, with type II antibodies potentially being superior to type I in the depletion of tissue-infiltrating B cells

Brain Shuttle Neprilysin reduces central Amyloid-β levels.

Reducing Amyloid β (Aβ) in the brain is of fundamental importance for advancing the therapeutics for Alzheimer`s disease. The endogenous metallopeptidase neprilysin (NEP) has been identified as one of the key Aβ-degrading enzymes. Delivery of NEP to the brain by utilizing the Brain Shuttle (BS) transport system offers a promising approach for clearing central Aβ. We fused the extracellular catalytic domain of NEP to an active or inactive BS module. The two BS-NEP constructs were used to investigate the pharmacokinetic/pharmacodynamics relationships in the blood and the cerebrospinal fluid (CSF) in dose-response and multiple dosing. As previously shown, NEP was highly effective at degrading Aβ in blood but not in the CSF compartment after systemic administration. In contrast, the NEP with an active BS module led to a significant CSF exposure of BS-NEP, followed by substantial Aβ reduction in CSF and brain parenchyma. Our data show that a BS module against the transferrin receptor facilitates the transport of an Aβ degrading enzyme across the blood-brain barriers to efficiently reduce Aβ levels in both CSF and brain

Obinutuzumab-Induced B Cell Depletion Reduces Spinal Cord Pathology in a CD20 Double Transgenic Mouse Model of Multiple Sclerosis

B cell-depleting therapies have recently proven to be clinically highly successful in the treatment of multiple sclerosis (MS). This study aimed to determine the effects of the novel type II anti-human CD20 (huCD20) monoclonal antibody (mAb) obinutuzumab (OBZ) on spinal cord degeneration in a B cell-dependent mouse model of MS. Double transgenic huCD20xHIGR3 (CD20dbtg) mice, which express human CD20, were immunised with the myelin fusion protein MP4 to induce experimental autoimmune encephalomyelitis (EAE). Both light and electron microscopy were used to assess myelination and axonal pathology in mice treated with OBZ during chronic EAE. Furthermore, the effects of the already established murine anti-CD20 antibody 18B12 were assessed in C57BL/6 wild-type (wt) mice. In both models (18B12/wt and OBZ/CD20dbtg) anti-CD20 treatment significantly diminished the extent of spinal cord pathology. While 18B12 treatment mainly reduced the extent of axonal pathology, a significant decrease in demyelination and increase in remyelination were additionally observed in OBZ-treated mice. Hence, the data suggest that OBZ could have neuroprotective effects on the CNS, setting the drug apart from the currently available type I anti-CD20 antibodies

Differences in expression levels of surface receptors between the cultured BECs and freshly isolated BECs.

<p>The RPL4 normalized mouse expression values (Mouse) (RPL4 normalized) are compared to the also RPL4 normalized expression values of hpBECs (Average P) and hCMEC/D3 (Average D3). The lower graph shows the absolute expression levels for each cell type. The upper graph shows the ratio between RPL4 normalized mouse BECs and hpBECs (Ratio M/P), the ratio between RPL4 normalized mouse BECs and hCMEC/D3 (Ratio M/D3) and the ratio between hpBECs and hCMEC/D3 (Ratio P/D3). Four genes that are expressed in much lower levels in both the hpBECs and the hCMEC/D3 cell line are Lrp8, IR, IGF1R and FcRn.</p

Differences in expression levels of SLC transporter between cultured BECs and freshly isolated BECs.

<p>The RPL4 normalized mouse expression values (Mouse) are compared to the also RPL4 normalized expression values of hpBECs (Average P) and hCMEC/D3 (Average D3). The lower graph shows the absolute expression levels for each cell type. The upper graph shows the ratio between RPL4 normalized mouse BECs and hpBECs (Ratio M/P), the ratio between RPL4 normalized mouse BECs and hCMEC/D3 (Ratio M/D3) and the ratio between hpBECs and hCMEC/D3 (Ratio P/D3). For instance genes that are expressed in much lower levels in both hpBECs and the hCMEC/D3 cell line are GLUT1, MCT8 and OAT3.</p

Immortalization influences BEC phenotype, growth behavior and expression of cell division related genes.

<p><i>(</i><b><i>A–C</i></b><i>)</i> Flow cytometry analysis of confluent hCMEC/D3 (red population, red histograms) and hpBECs (blue population, blue histograms) seeded on collagen I coated inserts. Staining was done with indicated antibodies or relevant isotype controls (grey population, tinted histograms). The average population sizes and standard deviations of three similar experiments are shown in <b>A</b>, whereas one representative experiment of three is shown in <b><i>B & C</i></b> (MFI = mean fluorescence intensity). <i>(</i><b><i>D</i></b><i>)</i> Real time monitoring of adherent BECs cultured in Resting (RM) or Growth Medium (GM) by the xCeLLIgence System. The curves show the time-, attachment- and density-dependent cell growth and viability of the individual BEC lines respectively culturing conditions. <i>(</i><b><i>E</i></b><i>)</i> Comparison of gene expression between hCMEC/D3 cells and hpBECs. The data is represented as a dot plot on a log₂ scale, where each point represents a probe set on the gene chip. Red and blue dots indicate probe sets, which have higher expression in hCMEC/D3 (red) or higher expression in hpBECs (blue). The mean expression values are averaged expression values for both cell lines. <i>(</i><b><i>F</i></b><i>)</i> The enrichment map displays the differently expressed gene sets between the two BEC lines. Red node color represents higher expression in hCMEC/D3 cells, whereas blue represents higher expression in hpBECs. Node size is proportional to the number of genes in the gene set and edge thickness represents the degree of overlap between two gene sets. Labels for the clusters of functionally related gene sets were manually assigned: 1) Mitosis, 2) DNA Repair, 3) Anaphase Promoting Complex, 4) Immune and Virus Response, 5) RNA Processing, 6) Cell-Cell Adhesion, 7) Differentiation/Maturation/Development, 8) Metabolic Processes, 9) Miscellaneous.</p

Differences in expression levels of ABC transporter between cultured BECs and freshly isolated BECs.

<p>The RPL4 normalized mouse expression values (Mouse) are compared to the also RPL4 normalized expression values of hpBECs (Average P) and hCMEC/D3 (Average D3). The lower graph shows the absolute expression levels for each cell type. The upper graph shows the ratio between RPL4 normalized mouse BECs and hpBECs (Ratio M/P), the ratio between RPL4 normalized mouse BECs and hCMEC/D3 (Ratio M/D3) and the ratio between hpBECs and hCMEC/D3 (Ratio P/D3). Three genes that are expressed in much lower levels in both the hpBECs and the hCMEC/D3 cell line are Pgp1, MRP4 and MDRA1.</p