Effects of Hawthorn Crataegus Plant
Extracts on Endothelial Cells
and Cardiomyocytes
By Linda Phan
Mentor: Dr. Theresa Filtz
Congestive Heart Failure
:disorder where the heart loses the ability to pump
blood efficiently
4.5 million patients in US
400,000 diagnosed each
year
Source: National Institute of Health
Hawthorn Crataegus laevigata
Extracts = Botanical
medicine
Alleviates symptoms of
congestive heart failure
Increases exercise tolerance
Active ingredients?
Increases blood flow out of the
heart
Affects blood pressure
Congestive Heart Failure
Causes:
Hypertension = high blood
pressure
Hypertrophy = thickening
of tissue, caused by increased
cell size
My Summer…
Methods Development
Endothelial Cells- Project #1
• Hypertension = high blood
pressure
Cardiomyocytes - Project #2.
w/Dr. Shannon Long
Project #1: Endothelial Cells
Endothelial Cells
Nitric Oxide
(NO)
Relaxes Smooth Muscle
Relaxed muscle disables contraction = dilation
How does NO relax smooth muscle?
NO

Inactivates
contraction
Dephosphorylated
Mechanism of smooth muscle relaxation
Does Hawthorn extract affect NO release
in endothelial cells?
Quantify NO release
using assays
1. Develop assays able
to produce consistent
results
EA.hy926 cells in culture
From the Human Umbilical Vein
2. Then observe
Hawthorn’s effects
Nitric Oxide Production
Mechanism of NO production in an endothelial cell
Radiotracer Assay’s Mechanism
14C-Arginine
 14C-Citrulline
Measure 14C-Citrulline
product
Correlate with NO
production
Does the protocol work?
EA.hy926 Cells
with 14C-Arginine
DMSO
(-)
A23187
(+)
A23197 – calcium ionophore, increases
intracellular [Ca2+] by increasing cell permeability
Nitric Oxide Production
Mechanism of NO production in an endothelial cell
Edited diagram by Cayman Chemical
Results
DMSO vs. A23187
0.00000007
0.00000006
0.00000005
14C0.00000004
Citrulline
0.00000003
(m m ol)
0.00000002
0.00000001
0
1
2
Trial
3
DMSO (-)
A23187 (+)
Results
Percentage Difference
between (+) and (-) controls
80
70
60
50
40
30
20
10
0
1
2
Trials
3
What’s next?
Achieve consistency.
How reliable is
Dowex resin?
Is resin holding the
unused C-14Arginine?
Dowex Resin
Ion-exchange
Resin overloaded?
Thin Layer Chromatography
Coming Soon!
Before Dowex
After Dowex
My Summer…
Methods Development
Endothelial Cells- Project #1
Cardiomyocytes - Project #2,
w/Dr. Shannon Long
• Hypertrophy = thickening of tissue,
caused by increased cell size
Project #2: Cardiomyocytes
Heart muscle cells
From mice pups
Contracts in media
Contracted vs. Relaxed
Contracted
Contracted vs. Relaxed
Relaxed
Contracted vs. Relaxed
Relaxed
Contracted vs. Relaxed
Contracted
Have already found …
Epinephrine
Beating
cells
Have already found…
Epinephrine
NONBeating
cells
Hypertrophy
: thickening of tissue, caused by increased cell size
1.“Create” hypertrophy in
cardiomyocytes
•
Phenylephrine
2. Hypertrophy?
3. Treat with Hawthorn on cells with
hypertrophy, observe. (coming soon)
Does Phenylephrine induce hypertrophy?
Cardiomyocytes
Untreated
(Control)
PBS
(-)
Phenylephrine
(+)
Results
Average Cell Area
4100
3900
3700
3500
Untreated
Cell Area
3300
(pixels)
3100
PBS (-)
Phenylephrine (+)
2900
2700
2500
Treatments
What’s Next?
Collect more data.
If hypertrophy in cells is successful, treat
with Hawthorn.
Dr. Filtz’s Long-Term Goals
Define the active components in Hawthorn with
cardiovascular activity.
Fractionation of the extracts to identify active
components.
Finally, determine proper dosing and toxic
effects.
Acknowledgements
Howard Hughes Medical Institute (HHMI)
Undergraduate Research, Innovation, Science, and
Creativity (URISC)
Dr. Theresa Filtz
Dr. Shannon Long
The Filtz Lab
Dr. Kevin Ahern
Thank you for your time!