Human Brain Microvascular Endothelial Cells and BBB

Fc-saxatilin used to Modulate Blood-Brain Barrier (BBB)
Highlights
•Fc-saxatilin prevents VEGF-induced permeability in Neuromics' human brain microvascular cells (HBMECs).
 •Fc-saxatilin inhibits VEGF-induced Src and Fak phosphorylation in HBMECs.
 •Fc-saxatilin blocks the downregulation of claudin-5 expression by VEGF in HBMECS.

Hyun-Jung Choi, Na-Eun Kim, Il Kwon, Dukhwan Choi, Jayoung Kim, Ji Hoe Heo. (2019). Fc-saxatilin inhibits VEGF-induced permeability by regulating claudin-5 expression in human brain microvascular endothelial cells. Microvascular Research. DOI: https://doi.org/10.1016/j.mvr.2019.103953

Figure: Fc–saxatilin attenuates the phosphorylation of Fak induced by VEGF. A) HBMECs were treated with pretreated with vehicle (PBS, 0) or Fc-saxatilin (50, 100, or 300 ng/ml) in the basal medium (0.5% FBS) for 10 min and treated with VEGF (100 ng/ml) for 1 h. Control cells were incubated in the control medium without Fc-saxatilin and VEGF. Cell lysates were subjected to an immunoblot analysis with antibodies against either phospho-Fak, Fak, or actin, as indicated. Representative data from three separate experiments are shown. B) Bands in the immunoblots were quantified using ImageJ and normalized to actin (n = 3); *P < 0.05 compared to the vehicle only-treated control or VEGF-treated control.

These cells are also used to formulate our 3-D Blood-Brain Barrier Model.

Sex Differences on the Behavior of Human Brain Microvascular Endothelial Cells (HBMECS)

The Risk of Stroke
There are sex differences in risk for stroke and small vessel ischemic disease in the brain. Microvesicles (MV) derived from activated cells vary by cell of origin and the stimulus initiating their release. MV released from cells activated by inflammatory and thrombotic factors have the potential to disrupt endothelial cells of the brain microvasculature. Therefore, experiments were designed to identify sex differences in the phenotype of MV released from cultured human brain microvascular endothelial cells (HBMEC) in response to inflammatory and thrombotic stimuli (Biology of Sex Differences201910:26 https://doi.org/10.1186/s13293-019-0241-y).

Neuromics' HBMECS, derived from a male donor, were used to study the differences between Male and Female Cells. There are indeed striking differences in the expression of Cell Adhesion Molecules when the male and female cells were stimulated with inflammatory and thrombotic factors.

4 Fold increase in release of MV from male (open bars) and female (black bars) HBMEC expressing cell adhesion molecules following stimulation with either TNFα (20 ng/ml) or THR (2 U/ml) for 20 h

These molecules, in part, control tight junctions in HBMECS. In stroke patients, these are disrupted resulting in injury to the brain.

The findings  underscore the appropriateness of identifying the sex of cells used in research studies of MV disposition within the vasculature, as well as the sex of cells used to generate MV for in vitro studies

Human Primary Cells Trifecta

Tri-culture of Neuromics' Human Primary Cells in 3-D BBB Model

I mentioned in an earlier post that we take Researchers questioning the validity of our Human Primary Cells very seriously. We follow up with our clients to make sure our cells are working as expected. If they are not 100% happy, we offer a free replacement or full refund.

...So it is encouraging to see Researchers using our Human Pericytes,  Brain Microvascular Endothelial Cells (HBMES) and Astrocytes to build a static 3-D Blood-Brain Barrier Model.

Ece Bayir, M. Mert Celtikoglu, Aylin Sendemira. The use of bacterial cellulose as a basement membrane improves the plausibility of the static in vitro blood-brain barrier model. https://doi.org/10.1016/j.ijbiomac.2018.12.257.

Image: Pericytes, HBMECS, and Astrocytes 5 days after culturing. Live cells (green) and dead cells (red).

The investigators concluded, "Caffeine and sucrose permeability values obtained from all models were close to literature data and physiological values."

Human Brain Microvascular Endothelial Cell in Action

Recent Pubs
Our Human Brain Microvascular Endothelial Cells (HBMECs) are known for the performance in drug discovery and tox assays. These 21-CFR compliant cells are used in the "blood side" of our 3-D BBB Models.

1. Shavali Shaik, Bridget Kennis, Shinji Maegawa, Keri Schadler, Yang Yanwen, Keri Callegari, Rishi R. Lulla, Stewart Goldman, Javad Nazarian, Veena Rajaram, Jason Fangusaro, and Vidya Gopalakrishnan. (2018). REST upregulates gremlin to modulate diffuse intrinsic pontine glioma vasculature. Oncotarget. 2018 Jan 12; 9(4): 5233–5250. doi: 10.18632/oncotarget.23750 
2. Hu et al. (2016). Epigenetic Activation of WNT5A Drives Glioblastoma Stem Cell Differentiation and Invasive Growth. Cell. 167, 1281–1295. http://dx.doi.org/10.1016/j.cell.2016.10.039,
   

GREM-1 is required for tube formation in vitro (A) Q-RT-PCR analysis of GREM-1 gene expression in SU-DIPG-IV cells stably expressing either control shRNA or GREM-1-specific shRNA. Lentiviral constructs expressing two different GREM-1 shRNAs (GREM-1.1 and GREM-1.2) were used to knockdown GREM-1. Efficiency of GREM-1 knockdown was determined by Q-RT-PCR and expression was normalized to 18s RNA. Significance is as shown (**less than.01). (B-C) HUVEC or human brain microvascular endothelial cells (HBMEC) were cultured in endothelial cell medium and or conditioned medium from either control shRNA or shGREM-1.2 transfected SU-DIPG-IV cells. Tube formation in matrigel was measured after 16h and images were obtained. The ability of GREM-1 to rescue loss of tube formation upon REST knockdown was determined by addition of human-recombinant GREM-1 (rGREM-1) to conditioned media-endothelial media mix. Scale bars, 100μm. (C) Quantification of tubes in matrigel shown in Figure B (right panels). Data shown is mean +/- SD, ***p less than .001, n=3. (D) Western blot analysis to assess VEGFR2 levels in SU-DIPG-IV, -VI and –XIII cells, HUVECs and HBMECs was done using anti-VEGFR2 antibodies. Tubulin served as a loading control. (E) Western blot analysis was performed to assess AKT signaling downstream of GREM-1 interaction with its potential receptor VEGFR2 in HUVEC and HBMEC. Anti-pAKT (S473), anti-pAKT (T308), total AKT, and anti-actin were employed.

If these cells fit your assay requirements and need more data/info, do not hesitate to contact me, Pete Shuster, CEO at pshuster@neuromics.com or cell: 612-801-1007.

Human Endothelial Cells and Drug Discovery

Pure, Potent and Culture Ready
Alterations of endothelial cells and the vasculature play a central role in the pathogenesis of a broad spectrum of the most dreadful of human diseases, as endothelial cells have the key function of participating in the maintenance of patent and functional capillaries. This makes them a key component for Drug Discovery.

We are proud that Biotech and Biopharma Cos. are increasingly using our cells. We believe we represent the best options. Here're some of the cells currently available:

• Human Bladder Microvascular Endothelial Cells
• Human Cardiac Microvascular Endothelial Cells
• Human Dermal Lymphatic Microvascular Endothelial Cells
• Human Dermal Microvascular Endothelial Cells
• Human Glomerular Microvascular Endothelial Cells
• Human Large Intestine Microvascular Endothelial Cells
• Human Ovarian Microvascular Endothelial Cells
• Human Pancreatic Microvascular Endothelial Cells
• Human Prostate Microvascular Endothelial Cells
• Human Retinal Microvascular Endothelial Cells
• Human Synovial Microvascular Endothelial Cells
• Human Tonsil Microvascular Endothelial Cells

Microvascular Endothelial Cells in Action

Figures: HRMECs treated with SUZ12 siRNA demonstrate increased miR-200b expression, decreased VEGF expression and decreased endothelial branching. A,B: Gene knockdown of EZH2 and SUZ12 was confirmed by qPCR. C,D: In HRMECs transfected with EZH2 siRNA in HG, miR-200b and VEGF were not significantly different from HG+control siRNA but decreased compared to NG+control siRNA. In HRMECs transfected with SUZ12 siRNA in HG, miR-200b was significantly increased with decreased levels of VEGF compared to HRMECs transfected with control siRNA, with levels similar to NG+control siRNA. E,F: Tube formation assay to measure endothelial branching. HRMECs transfected with HG+control siRNA demonstrated significantly increased branching compared to NG+control siRNA. Transfection of EZH2 siRNA did not reduce endothelial branching significantly compared to HG+control siRNA. However, transfection of SUZ12 siRNA significantly reduced endothelial branching compared to HG+control siRNA. 10.1371/journal.pone.0123987
We will keep you posted as we add more cell options.

New Human Brain Microvascular Cells

HBMECS for BBB Researchers

I am pleased by the growing positive response to our Human Endothelial Cells offerings. Our Human Brain Microvascular Endothelial Cells have, far and away, been the most popular. We want to make it easier for you to "buy and try" We are offering them for only 699 USD/500,000 Cells through Aug. 31st, 2015. Note: customers have passaged these cells up to 15 X.

Image: Human Brain Microvascular Endothelial Cells in Culture.

Questions? Please contact me: pshuster@neuromics.com or 612-801-1007.