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Effects of Hawthorn Crataegus Plant Extracts on Endothelial Cells
and Cardiomyocytes
By Linda Phan
Mentor: Dr. Theresa Filtz
Congestive Heart Failure
4.5 million patients in US
400,000 diagnosed each year
Source: National Institute of Health
:disorder where the heart loses the ability to pump blood efficiently
Hawthorn Crataegus laevigata
Extracts = Botanical medicine
Alleviates symptoms of congestive heart failure
Increases exercise tolerance
Increases blood flow out of the heart
Affects blood pressure
Active ingredients?
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?
Mechanism of smooth muscle relaxation
NO
Inactivates contraction Dephosphorylated
Does Hawthorn extract affect NO release in endothelial cells?
Quantify NO release using assays
1. Develop assays able to produce consistent results
2. Then observe Hawthorn’s effects
EA.hy926 cells in culture
From the Human Umbilical Vein
Nitric Oxide Production
Mechanism of NO production in an endothelial cell
Radiotracer Assay’s Mechanism
Measure 14C-Citrulline product
Correlate with NO production
14C-Arginine 14C-Citrulline
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
00.000000010.000000020.000000030.000000040.000000050.000000060.00000007
14C-Citrulline (mmol)
1 2 3
Trial
DMSO vs. A23187
DMSO (-)
A23187 (+)
Results
01020304050607080
1 2 3
Trials
Percentage Difference between (+) and (-) controls
What’s next?
Achieve consistency.
How reliable is Dowex resin?
Is resin holding the unused C-14-Arginine?
Dowex Resin
Ion-exchange
Resin overloaded?
Thin Layer ChromatographyComing 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 …
Beating cells
Epinephrine
Have already found…
NON-Beating
cells
Epinephrine
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)
Phenylephrine
(+)
PBS
(-)
Results
Average Cell Area
2500
2700
2900
3100
3300
3500
3700
3900
4100
Treatments
Cell Area (pixels)
Untreated
PBS (-)
Phenylephrine (+)
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!