Heart Disease In 1995: 481,000 deaths related to Coronary Artery Disease (CAD)

1. Heart Disease In 1995: • 481,000 deaths related to Coronary Artery Disease (CAD) • 1,100,000 new or recurrent cases of CAD • Estimated that 7.2 million people experienced angina to some degree Treatment • 434,000 angioplasties performed • 573,000 Bypasses performed • 60,000-100,000 patients not good candidates for bypass/angioplasty (Possibly up to 250,000 patients a year)

2. Use of Vascular Endothelial Growth Factor (VEGF) as a Treatment for End Stage Coronary Artery Disease (CAD) By: Jeremy Gillis Senior…Biochemistry and Molecular Biology

3. Current Treatments for CAD • Percutaneous Transluminal Coronary Angioplasty or PTCA (434,000) • Coronary Artery Bypass Graft (CABG) “cabbage” (573,000) • Vascular Stents (wire props for an artery) • Rotational Atherectomy (much like a drill)

4. Problems with Current Treatments • Restenosis • Graft disease • Arterial puncture • Coronary thrombosis How can we help people who don’t respond well or are not good candidates for conventional treatments?

5. It is thought that VEGF is involved in Angiogenesis Angiogenesis: the formation of new blood vessels (collaterals) from existing microvessels Contributes to the preservation of ischemic tissue and myocardial pump function after myocardial infarction Important in: • Embryogenesis (called vasculogenesis) • Wound healing • Tumor growth and metastasization • Rheumatoid arthritis • Ischemic heart disease • Ischemic peripheral vascular disease

6. Inducing Angiogenesis 1. Need a stimulus Hypoxic tissue, Ischemic tissue, Mechanically damaged tissue 2. Need expression of angiogenic molecules to initiate angiogenesis VEGF, FGF, TGF, PDGF 3. Need angiogenesis to occur 1. Proliferation and migration of endothelial cells from the microvasculature 2. Controlled expression of proteolytic enzymes 3. Breakdown and reassembly of extracellular matrix 4. Morphogenic process of endothelial tube formation Mechanism of Angiogenesis not completely known

7. Why use VEGF to Promote Angiogenesis? VEGF (vascular endothelial growth factor) • Specific for only endothelial cells • May inhibit smooth muscle growth…reduce restenosis FGF (fibroblast growth factor) • Associated with tumor angiogenesis • Can stimulate growth in other cells besides endothelial cells • Not as specific as VEGF TGF- (transforming growth factor ß) • Indirect angiogenesis effect • Possibly induces VEGF expression (Protein Kinase C pathway) PDGF (platelet derived growth factor) • Not well characterized in angiogenesis

8. Other VEGF Characteristics • VEGF expressed by Macrophages, fibroblasts, smooth muscle cells, endothelial cells (all are present in the heart) • Action is direct because of the exclusive specificity for receptors (flt-1 and flk-1) • Receptors only found on endothelial cells • Causes activation of many other genes involved in angiogenic response

9. How to Deliver VEGF Protein Therapy • Direct injection of protein • Time delay delivery • Local intercoronary bolus Gene Therapy Adenovirus vector • Excellent specificity for endothelial cells • Extended expression of VEGF Direct gene transfer • Involves direct injection of eukaryotic plasmid DNA containing VEGF cDNA Should VEGF administration prove effective, it is likely that VEGF/VEGF DNA will be delivered on a catheter platform

10. Case Studies Injection of naked VEGF cDNA contained in an Eukaryotic Expression Vector Jeffery Isner et al. St. Elizabeth’s Medical Center Phase I clinical trial…designed to assess safety and bioactivity of treatment methods Limited sample…only 5 patients involved • Prior Bypass and/or angioplasty • Class 3-4 Angina • No longer respond to additional treatment

11. Results

12. Also notable: • Nitroglycerin usage dropped from 7.7 pills per day to 1.4 per day for the group (60 days post) • Effective biological outcomes despite low transfection rates • Because of the condition of the patients in the study, the improvements to health were not likely random events All 5 patients had remarkable gains in quality of life post procedure

14. Animal Data: • Charles Mack et al. New York Hospital-Cornell Medical Center • Administration VEGF gene through Adenovirus mediated gene therapy • Preclinical work to determine efficacy in an animal model of ischemia

16. Model: • Pig with a constrictor band around circumflex artery to induce myocardial infarction and ischemia • Eventually results in complete occlusion of circumflex artery Vector: • Adenovirus vector in E1a-, partial E1b-, and partial E3- mutations (makes them replication deficient) • Adenovirus used because of the natural selectivity for endothelial cells • Minimal inflammation detected in animals 4 weeks post therapy • In vivo conformation of expression confirmed by ELISA 3 days after injection

17. Results • Treatment Resulted in significantly reduced ischemic area (area of oxygen starved tissue) and • Ischemic maximum (severity of ischemia) in treated animals • Strength of heartbeat returned in treated animals more than untreated animals • More vessels visible angiographically in treated animals vs. untreated animals • Treated animals seemed to route around the occlusion as demonstrated by the filling of branching arteries

18. Why it works? • Placebo effect? • VEGF stimulates growth of “collateral” vessels? • Microvessel growth due to physical damage of heart? • Real or perceived Angiogenesis?

19. Problems: • Doesn’t work as well on older patients with more advanced disease • VEGF may stimulate undetected cancer growth (tumors cannot be larger than a few mm3 without revascularization? • Limited number of trials and patients • Treatment kills some patients? • What are the effects on women? • No placebo substance given for ethical reasons

20. References Battegay, E.J. Angiogenesis: mechanistic insights, neovascular diseases, and therapeutic prospects. Journal of Molecular Medicine (1995) 73:333-346. Losordo, Douglas W., et al. Gene therapy for Myocardial Angiogenesis: Initial Clinical Results With Direct Myocardial Injection of phVEGF165 as Sole Therapy for Myocardial Ischemia. Circulation (1998) 98:2800-2804. Mack, Charles A., et al. Biologic Bypass With the Use of Adenovirus-Mediated Gene Transfer of the Complementary Deoxyribonucleic Acid for Vascular Endothelial Growth Factor 121 Improves Myocardial Perfusion and Function in the Ischemic Porcine Heart. Journal of Thoracic and Cardiovascular Surgery (1998) 115:168-77. Li, Jian, et al. Stretch-induced VEGF Expression in the Heart. Journal of Clinical Investigation (1997) 100:18-24. Seko, Yoshinori, et al. Serum levels of vascular endothelial growth factor in patients with acute myocardial infarction undergoing reperfusion therapy. Clinical Science (1997) 92:453-454. Lopez, John J., et al. VEGF administration in chronic myocardial ischemia in pigs. Cardiovascular Research (1998) 40:272-281. Metais, Caroline, et al. Effects of coronary artery disease on expression and microvascular response to VEGF. American Journal of Physiology (1998) 275:H1411-H1418.