1. Effects of Hawthorn Crataegus Plant Extracts on Endothelial Cells and Cardiomyocytes By Linda Phan Mentor: Dr. Theresa Filtz

2. 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

3. 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?

4. Congestive Heart Failure Causes: Hypertension = high blood pressure Hypertrophy = thickening of tissue, caused by increased cell size

5. My Summer… Methods Development Endothelial Cells- Project #1 Hypertension = high blood pressure Cardiomyocytes - Project #2. w/Dr. Shannon Long

6. Project #1: Endothelial Cells Endothelial Cells Nitric Oxide (NO) Relaxes Smooth Muscle Relaxed muscle disables contraction = dilation

7. How does NO relax smooth muscle? Mechanism of smooth muscle relaxation NO  Inactivates contraction Dephosphorylated

8. 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

9. Nitric Oxide Production Mechanism of NO production in an endothelial cell

10. Radiotracer Assay’s Mechanism Measure 14 C-Citrulline product Correlate with NO production 14 C-Arginine  14 C-Citrulline

11. Does the protocol work? EA.hy926 Cells with 14 C-Arginine DMSO (-) A23187 (+) A23197 – calcium ionophore, increases intracellular [Ca 2+ ] by increasing cell permeability

12. Nitric Oxide Production Mechanism of NO production in an endothelial cell

13. Edited diagram by Cayman Chemical

14. Results

16. What’s next? Achieve consistency. How reliable is Dowex resin? Is resin holding the unused C-14- Arginine?

17. Dowex Resin Ion-exchange Resin overloaded?

18. Thin Layer Chromatography Coming Soon! Before DowexAfter Dowex

19. My Summer… Methods Development Endothelial Cells- Project #1 Cardiomyocytes - Project #2, w/Dr. Shannon Long Hypertrophy = thickening of tissue, caused by increased cell size

20. Project #2: Cardiomyocytes Heart muscle cells From mice pups Contracts in media

21. Contracted vs. Relaxed Contracted

22. Contracted vs. Relaxed Relaxed

23. Contracted vs. Relaxed Relaxed

24. Contracted vs. Relaxed Contracted

25. Have already found … Beating cells Epinephrine

26. Have already found… NON- Beating cells Epinephrine

27. 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)

28. Does Phenylephrine induce hypertrophy? Cardiomyocytes Untreated (Control) PBS (-) Phenylephrine (+)

29. Results

30. What’s Next? Collect more data. If hypertrophy in cells is successful, treat with Hawthorn.

31. 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.

32. 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!