ABRI PRESENTATION 7.23.14.pptx
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Transcript of ABRI PRESENTATION 7.23.14.pptx
Wnt signaling pathway at the BBB:
a potential target for drug delivery and stroke-induced vascular leakage
A foundational study byTaylor R. Liles
Pharmaceutical Sciences intern under Dr. Abraham Al Ahmad
The Blood Brain Barrier
• Physical barrier:
• Endothelial cells, Pericytes, Astrocytes, and neurons
• TJ Complexes
• Chemical barrier:
• Nutrients transporters (glucose and amino acids)
• RMTs for hormones (insulin)
• Drug efflux pumps (Pgp, BCRP…)
http://www.nature.com/nrd/journal/v6/n8/full/nrd2368.html
Hypoxia Inducible factor 1
• Heterodimeric transcription factors
• O2 dependant α-
subunit
• bio-mechanisms altered via target gene activation
• “survival mode” http://www.nature.com/nrc/journal/v2/n1/fig_tab/nrc704_F1.html
The Wnt pathway
• Mamillian genetic (Wnt, Dsh, Frzz)
• Canonical Wnt pathway (β-Catenin production)
• "Body axis patterning" in early embryonic development
• Implicated in BBB differentiation (Al Ahmad, et. Al 2012)
http://www.wormbook.org/chapters/www_wntsignaling/wntsignaling.html
Objectives
• Does altering Wnt ligands affect efflux transporters in the BBB?
• Can this effect be used to modulate the HIF-1 pathway during an ischemic event?
• Provide a proof of concept in HeLa cells to be carried on.
Methods
• Cultured samples of HeLa cells
• Control, CHIR, XAV
• Normoxic, Hypoxic, Hypoxic-reperfusion
• Accumulation protein
assay
• Western Blotting
• Immunochemistry
Hypoxia did not affect HeLa cell
viability
Normoxia Hypoxia Reoxygenation
Brig
htfie
ld
Normoxia (21% O2)
Hypoxia (1% O2)
24 hours
5 hours
Reoxygenation (21% O2)
Effects of hypoxia on Pgp and
BCRP activity in HeLa cells
Pgp BCRP
Control +CHIR99021
+XAV939
Bri
ghtf
ield
GLU
T1
Effect of Wnt modulation on HeLa cell viability and GLUT1 expression
Con
trol
+CH
IR99
021
+XA
V93
9P-gp
Effect of Wnt on Pgp and BCRP functional activity
Pgp
BCRP
Wnt inhibition reduces functional Pgp activity
in HeLa cells
Con
trol
+CH
IR99
021
+XA
V93
9
HIF-1α
Wnt activation increases HIF-1 stabilization under normoxia
Summary
Discussion
• Hela Cells respond to Wnt pathway modulators
• Hypoxia increases PGP activity
• Wnt activation increased PGP and GlUT1 but not BCRP
• Wnt appears to interact with the HIF-1α pathway to increase PGP
Future Experiments
• Moving from HeLa cells to stem cell and BMEC samples
• Varying methods of Wnt modulation to see which ligands are involved
Acknowledgements
• Dr. Abraham Al-Ahmad (mentor)
• Nihar Kinarivala (grad student)
• Teresa Carlisle
• ABRI Luncheon presenters