1. Heart Disease In 1995: z481,000 deaths related to Coronary Artery Disease (CAD) z1,100,000 new or recurrent cases of CAD zEstimated that 7.2 million people experienced angina to some degree Treatment z434,000 angioplasties performed z573,000 Bypasses performed z60,000-100,000 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 zPercutaneous Transluminal Coronary Angioplasty or PTCA (434,000) zCoronary Artery Bypass Graft (CABG) “cabbage” (573,000) zVascular Stents (wire props for an artery) zRotational Atherectomy (much like a drill)

4. Problems with Current Treatments z Restenosis z Graft disease z Arterial puncture z 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: z Embryogenesis (called vasculogenesis) z Wound healing z Tumor growth and metastasization z Rheumatoid arthritis z Ischemic heart disease z 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) zSpecific for only endothelial cells zMay inhibit smooth muscle growth…reduce restenosis FGF (fibroblast growth factor) zAssociated with tumor angiogenesis zCan stimulate growth in other cells besides endothelial cells zNot as specific as VEGF TGF-  (transforming growth factor ß) zIndirect angiogenesis effect zPossibly induces VEGF expression (Protein Kinase C pathway) PDGF (platelet derived growth factor) zNot well characterized in angiogenesis

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

9. How to Deliver VEGF Protein Therapy z Direct injection of protein z Time delay delivery z Local intercoronary bolus Gene Therapy Adenovirus vector z Excellent specificity for endothelial cells z Extended expression of VEGF Direct gene transfer z 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 zPrior Bypass and/or angioplasty zClass 3-4 Angina zNo longer respond to additional treatment

11. Results

12. Also notable: zNitroglycerin usage dropped from 7.7 pills per day to 1.4 per day for the group (60 days post) zEffective biological outcomes despite low transfection rates zBecause 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: zCharles Mack et al. New York Hospital- Cornell Medical Center zAdministration VEGF gene through Adenovirus mediated gene therapy zPreclinical work to determine efficacy in an animal model of ischemia

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

17. Results zTreatment Resulted in significantly reduced ischemic area (area of oxygen starved tissue) and zIschemic maximum (severity of ischemia) in treated animals zStrength of heartbeat returned in treated animals more than untreated animals zMore vessels visible angiographically in treated animals vs. untreated animals zTreated animals seemed to route around the occlusion as demonstrated by the filling of branching arteries

18. Why it works? zPlacebo effect? zVEGF stimulates growth of “collateral” vessels? zMicrovessel growth due to physical damage of heart? zReal or perceived Angiogenesis?

19. Problems: zDoesn’t work as well on older patients with more advanced disease zVEGF may stimulate undetected cancer growth (tumors cannot be larger than a few mm 3 without revascularization? zLimited number of trials and patients zTreatment kills some patients? zWhat are the effects on women? zNo 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 phVEGF 165 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.