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