Effects of Hawthorn Crataegus Plant Extracts on Endothelial Cells and Cardiomyocytes By Linda Phan...

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!