1. Intramyocardial Bone Marrow Cell Injection for Chronic Myocardial Ischemia Leiden University Medical Center the Netherlands No conflicts of interest

2. Background Coronary artery disease is a major cause of morbidity and mortality. In many patients: progression of atherosclerosis  successive revascularization procedures  end-stage coronary anatomy ineligible for further conventional revascularization. A significant number of these patients has stress-induced ischemia resulting in angina pectoris despite maximal therapy.

3. Bone marrow-derived mononuclear cells Beeres et al, Heart 2008

4. BMC show a more favorable survival pattern Bone marrow-derived mononuclear cells Comparison with skeletal myoblasts and MSC Van der Bogt et al, circulation 2008;118[suppl 1]:S121–S129

5. Early phase human studies in patients with refractory angina* →Safe → Feasible → possibly beneficial effect on anginal symptoms myocardial perfusion global left ventricular function *Fuchs et al JACC 2003; Tse et al Lancet 04; Perin et al, circulation 2003 Human studies in refractory angina

6. Pilot study in Leiden Intramyocardial bone marrow cell injection 25 patients with chronic myocardial ischemia Results: –Intramyocardial bone marrow cell injection is safe –Improvements observed in: Myocardial perfusion Global LV function Anginal complaints Justified the initiation of a randomized, placebo-controlled, double-blind clinical trial Beeres et al, JNM 2006,47: 574-80

7. 2.8 mV3.6 mV 4.6 mV 6.5 mV 4.8 mV 3.2 mV * * * * * * * * * Baseline 3 months Region of interest: inferolateral Safety analysis: conduction

8. Bone marrow aspiration and isolation of mononuclear cells Bone marrow aspiration from iliac crest  80 ml under local anesthesia Isolation of mononuclear cells (Ficoll density gradient) 2.5 ml

9. Shaft Location sensor Tip electrode

10. Injection Procedure General region for treatment: ischemic area on SPECT NOGA map: target the specific treatment area by identifying viable myocardium within the ischemic region >= 7.9 mV <>= 11.0 % <<= 5.0 % <= 6.9 mV Maximum Unipolar Voltage (mV) Linear Local Shortening (%) stress HLA rest HLA

11. Inclusion criteria Severe refractory AP (CCS class 3-4) despite optimal medical care Ischemia on Tc-99m tetrofosmin SPECT No candidate for conventional revascularization (angiogram) Exclusion criteria Study design Heart failure symptoms and/or LV ejection fraction <35% Evidence of cancer Severe concurrent illness (eg. infection or renal failure)

12. Study design Primary outcome: Improvement in summed stress score (perfusion) as assessed by SPECT Other outcomes: –Efficacy: Myocardial perfusion at rest (SPECT) Global LV function (MRI) Anginal symptoms CCS class QOL (Seattle angina questionnaire) –Safety (arrhythmias, pericardial effusion, inflammation, myocardial damage)

13. Clinical Characteristics (n=50) Bone marrow cell group (n=25) Placebo group (n=25) P-value Age, years64±862±90.55 Gender (Male)23(92%)20(80%)0.41 Cardiovascular risk factors Smoking10(40%)12(48%)0.77 Hypertension12(48%)11(44%)1.00 Diabetes 13(52%)8(32%)0.25 Dyslipidemia12(48%)15(60%)0.57 Coronary artery disease in family16(64%)13(52%)0.56 BMI29±328±40.40 Current Medication Nitrates21(84%)21(84%)1.00 Beta-blockers24(96%)24(96%)1.00 Calcium channel blockers18(72%)18(72%)1.00 Statins25(100%)25(100%)1.00 ACE inhibitors19(76%)14(56%)0.23 History Prior Myocardial infarction14(56%)18(72%)0.37 Prior CABG24(96%)19(76%)0.10 Prior PCI16(64%)13(52%)0.57

14. Safety: events at 12 months Periprocedural –No myocardial infarction –No systemic inflammation –No arrhythmias –1 patient in placebo group: pericardial effusion > 5 mm –1 patient in the placebo group suffered from a cerebrovascular accident shortly after injection Follow-up –At 2.5 months: 1 death in cell group severe myocardial ischemia –At 2 months: 1 spondylodiscitis (S. Aureus) in placebogroup –At 3.5 months: 1 revascularization in cell group –At 8 months: 1 death in placebo group because of breast cancer –At 8 months: 1 stroke in cell group

15. Summed stress score (perfusion during exercise) 23.5 20.1 24.8 23.7 Myocardial perfusion (Tc99m-tetrofosmin SPECT)

16. Summed stress score (perfusion during exercise) 23.5 20.3 24.8 22.9 Myocardial perfusion (Tc99m-tetrofosmin SPECT) 21.0 23.5

17. Myocardial perfusion (Tc99m-tetrofosmin SPECT) Summed rest score (resting perfusion)

18. Myocardial perfusion (Tc99m-tetrofosmin SPECT) Summed rest score (resting perfusion)

19. Myocardial perfusion at 12 months follow-up 3.9 1.5 3.1 2.4 1.7 2.6

20. 54 53 57 56 LV function (gated SPECT) at 12 months follow-up

21. Quality of life at 12 months follow-up 55 64 69 70 64 57 61 62

22. CCS class at 12 months follow-up

23. Conclusion Safe and feasible procedure Significant effect of bone marrow cell injection on: –Myocardial perfusion –Global LV function –Anginal symptoms Effects on perfusion and clinical parameters are sustained at 12 months follow-up

24. Discussion Limitations –Current study not powered to assess the effect on prognosis –Long term effect not yet investigated Issues to be investigated –Long term effects –Effects of reinjection in patients with recurrent angina –Identification of responders/non-responders

25. Conclusion Autologous bone marrow-derived mononuclear cell injection in no-option patients with chronic myocardial ischemia is safe and is associated with a beneficial effect on anginal symptoms, myocardial perfusion and left ventricular function

26. Acknowledgements Molecular Cell Biology LUMC John van Tuyn Jim Swildens Twan de Vries IHB LUMC Vanessa van Zuylen Helene Roelofs Jaap Jan Zwaginga Wim Fibbe UMCU Pieter Doevendans Columbia University New York Mike Rosen Hannover Kai Wollert Berlin, Charite Carsten Tschoepe Cardiology LUMC Saskia Beeres Cheryl Dambrot Jan van Ramshorst Robert Grauss Melina den Haan Arti Ramkisoensing Niek Pluijmert Daniel Pijnappels Paul Steendijk Arnoud van der Laarse Jeroen Bax Martin Schalij Douwe Atsma Anatomy and Embryology LUMC Christian Freund Christine Mummery Nefrology LUMC Anton Jan van Zonneveld Ton Rabelink