Therapeutic Angiogenesis: Protein and Gene Therapies offer Hope to Patients with Myocardial Ischemia

1. Therapeutic Angiogenesis: Protein and Gene Therapies offer Hope to Patients with Myocardial Ischemia Ryan McAuley Dept. of Biology Furman University Greenville, SC

2. Palo Alto Veterans Affairs Healthcare System • Stanford University School of Medicine Department of Cardiology

3. Outline • Overview of Myocardial Ischemia: etiology, diagnosis, symptoms, and conventional treamtents • Angiogenesis: history and development • Overview of current studies • Results from clinical trials • Future Studies

4. Myocardial Ischemia • Gr. ischein “to hold back” + haima “blood” • Caused by: -Stenosis of coronary arteries -Acute blockage • Coronary blood flow inadequate for maintaining cardiac function • Result: cardiac muscle is deprived of essential nutrients and gas exchange • Symptoms: most common is angina pectoris

5. Diagnosis • Electrocardiogram (ECG) • Exercise Tolerance Test (ETT) • Myocardial Perfusion Imaging

6. Treatment • Complications include: myocardial infarction, cardiac arrhythmias, CHF, and low quality of life • Pharmacotherapy: combination of drugs -antiplatelet agents -antithrombotic drugs -lipid-lowering drugs -anti-anginal drugs • Invasive Therapies: -CABG and PCI

7. Limitations • Symptoms not relieved by drugs • Patient is not good candidate for invasive procedures

8. Angiogenesis • Offers hope to these “no-option” patients. • Current clinical trials to assess safety and efficacy for FDA approval • Definition: extension of already formed primitive blood vessels by budding of new capillaries through proliferation and migration of endothelial cells • Takes place during embryonic development and combined with vasculogenesis, is responsible for development of the circulatory system • Naturally-occurring process in adults that is prompted by hypoxia or ischemia after occlusion of an artery

9. Meet the Growth Factors • Fibroblast Growth Factor (FGF) -Peptide Family -Cross-species homology -Targets: endothelial cells, smooth muscle cells, fibroblasts, myocytes, and some tumor cells • Vascular Endothelial Growth Factor (VEGF) -Glycoprotein Family -Targets endothelial cells exclusively

10. The Process • Hypoxic conditions • VEGF upregulation occurs within 6 hours due to: -stabilization of mRNA coding -increased transcription due to activation of Hypoxia-Inducible Factor-1 (HIF-1) in the promoter region of VEGF • If this is a naturally-occurring process, why do pts. still have disabling chest pain??

11. The Problem • Animal studies have shown impaired angiogenesis and reduced endothelial cell viability in older animals • Decreased angiogenic activity also noted in diabetic and hypercholesterolemic mice • Since many patients. with myocardial ischemia have other health problems such as these, angiogenesis does not sufficiently improve coronary blood flow • Don’t be sad…

12. BE GLAD!!! • In all of these cases VEGF supplementation produced favorable results with regards to and Endothelial Cell Response Growth

13. Early Work • Discovered by Folkman in early 1970’s • Link between vascular GF’s and neovascularization associated with tumor growth • In 1983, Kumar et al. studied the presence of an “angiogenesis factor” in the human heart following MI • Mid-1980’s: several polypeptide growth factors associated with angiogenesis identified and purified • As a result, animal and human studies could be expanded • Pre-clinical animal studies used an ameroid constrictor to gradually occlude one of the coronary arteries. • Pigs, dogs, and rabbits have been used for models of therapeutic angiogenesis

14. Animal Studies • VEGF and FGF administered in various amounts and by different routes • Effectiveness measured by many means including: -size and number of vessels -measurement of coronary blood flow -quantitation of endothelial cell markers • Positive results for protein and genes, but a few problems

16. Current Studies • Endpoints • Gene Therapy Vs. Protein Therapy • Administration Route • Dosage -Placebo?

17. Common Endpoints • Change in total ETT time at baseline and after treatment • Frequency of angina: # of doses of nitro per week • Perfusion Imaging: MRI, angiography

18. ETT Evaluation

19. ST Segment

20. ST Segment Depression = Myocardial Ischemia Normal ST segment depression

21. Data Points from ETT • Time to onset of angina • Time to >1mm change in ST segment • Measurement of HR, BP, and ST segment depression at maximal exercise (angina pain rated as a 3 out of 4 or exhaustion) • Measurement of ST segment depression at 1, 3, and 5 minutes recovery

22. Overview of Current Studies • Comparisons: -Protein Therapy Vs. Gene Therapy -Administration Route -Dosage • FDA approval: -Phase I to determine safety/feasibility -All subsequent phases must include placebo group to determine efficacy

23. Physical Properties of GF • GOAL: High exposure to coronary vessels, Low systemic exposure • Protein Therapy: -recombinant form of FGF or VEGF • Gene Therapy: -VEGF or FGF inserted into a viral vector -Naked DNA plasmid encoding for transcription of VEGF or FGF

24. Characteristic Protein Therapy Gene Therapy Short-lived Prolonged More likely Less likely Easier Potential risk for inflammatory response if viral vector used No Yes Short-term, Long-term, but high level but low level Exposure to GF Need for repeat dose Readministration Exposure to foreign genetic material Systemic Exposure

25. Administration Routes • GOAL: Least invasive procedure that allows for Optimal uptake of GF’s • Many have been used • Most common are Intracoronary and Intramyocardial

26. Dosage • GOAL: Dose is Large enough to be effective in coronary arteries, but Small enough that systemic exposure is not a concern • Protein Therapy: µg/kg or ng/kg • Gene Therapy: -number of viral particles -DNA plasmids in units of µg • Escalating dose groups to determine how side effects and effectiveness are related to the amount of GF administered • Placebo group shows objective comparison to treatment group and randomization removes physician bias

27. Results of Phase I Studies • In all studies, favorable results were reported • Increased myocardial perfusion shown on MRI and angiography, increased ETT time compared to baseline, and decreased angina • However, small sample size, lack of placebo group, and nonrandomization result in poor predictive value

28. Rosengart et al. 1999 • n=21 • Randomized: No • Angiogenic Factor: VEGF121 viral vector • Administration Route: Intramyocardial • Results: Improved angiography results, increased exercise time, decreased angina

29. Results of Phase II Studies • Not consistently significant • Dramatically demonstrate normalizing effect of placebo group

30. Kleiman and Califf 2000FIRST multicenter study • n=337 total in 3:1 ratio of active agent to placebo • Randomized: Yes • Angiogenic Factor: FGF-2 • Administration Route: Intracoronary • Results: -No significant improvement in exercise time or stress nuclear perfusion imaging at 90 days -Less angina in treatment group (P=0.057) -Trend toward greater improvement in older and more symptomatic pts.

31. What Happened? • Small sample size • Insensitive end-points • Single-administration of GF • Acute myocardial ischemia in animal models Vs. chronic myocardial ischemia in humans

32. So… • While angiogenesis has great potential, more research needed • Short term goal: prove efficacy in large-scale, placebo-controlled trials • Determine long-term safety by addressing concerns…

33. Some Clinical Concerns • Cancer • Abnormal vascular growth in non-target tissues • Immune consequences of using viral vectors with foreign genetic material • Risks associated with local myocardial delivery • Note: these concerns have not all been validated in research and the list will most likely evolve in the future

34. The Future • Variations on the theme that increased exposure to GF’s yields optimal vascularization • Multiple doses and/or sustained release of recombinant proteins • Administration of multiple GF’s • Administration of HIF-1 • Autologous bone marrow injection

35. THE END Special thanks to Dr. Thompson for her support and guidance through this entire project. To Victor Froelicher, MD and Jonathan Myers, PhD for allowing me the opportunity to work with them and for their help with my paper. To my uncle Paul McAuley, PhD for the “referral” to the aforementioned Docs. To Soon-to-be-Dr. Schammel for her encouragement and technological assistance. To Dr. Turgeon for her help and enthusiasm. And finally, to Dean Charles Brock, PhD for allowing Furman students the opportunity to participate in internships such as these through Furman Advantage funding.