1. Comparative Progression of Atherosclerosis in ApoE-/- Mice Exposed to ETS and CAPs Lung Chi Chen, Ph. D. Department of Environmental Medicine NYU School of Medicine 7th ICCAD, Venice Italy Oct. 10, 2007

2. Health Risks of Environmental Tobacco Smoke (ETS) Exposure u ETS increases coronary heart disease (CHD) in non-smokers by a factor of 1.3. u Long term exposure to ETS caused 40,000 - 60,000 excess heart disease deaths in the US. u Smoking produces atherogenic serum lipid profile u Smoking increases intima-media thickness coronary progression

3. When Pollution Gets High, Deaths Increase: 1952 London Fog Episode Deaths vs. PM

4. (Pope, Burnett, Thun, Calle, Krewski, Ito, and Thurston) (JAMA, 2002)

5. Association between Ambient Particle Pollution and Atherosclerosis Künzli et al, EHP 113, 201-206, 2005 In Los Angeles CIMT: carotid intima-media thickness

6. Schematic of the evolution of the atherosclerotic plaque Braunwald's Heart Disease: A Textbook of Cardiovascular Medicine, 7th ed

7. Common Mechanisms of Ambient Particles and Tobacco Smoke Induced Atherosclerosis Ambient Particles Cigarette Smoke Oxidative Stress (O 2 -, H 2 O 2, ONOO - ) Vascular Dysfunction, Prothrombotic Factors, Leukocyte, Platelet Activation, Lipid Peroxidation Adhesion Molecules, Smooth Muscle Proliferation Initiation and Progression of Atherothrombotic Diseases Inflammatory Gene Activation NO Regulation

8. EXPERIMENTAL DESIGN  ApoE -/- mice u Normal Chow (4% fat, w/w, 8% calories from fat) u High Fat Chow (21% fat, w/w, 42% calories from fat)  Exposed, whole body, to fine CAPs or filtered air for 6 hours/day, 5 days per week u May 29, - Dec. 7, 2007. u Average 6 hr Concentration: 165 µg/m 3 u equivalent to 30 ug/m 3 annual average.  Exposed, whole body, to side stream (SS) smoke or filtered air for 6 hours/day, 5 days per week, for 6 months u Average 6 hr Concentration: 450 µg/m 3 u Approximately equivalent to living with a smoker who smoke 3 cigarettes/day.

9. Ambient Particle Concentrating and Exposure Systems Animal Exposure Chambers VACES Modified DataScience ECG Receivers Inside of the exposure chambers

10. ETS Exposure Side-Stream Smoking Machine (CSM 2070, CH Tech) 2R4F, 2 sec 35 ml puff/min, 12 puffs/cigarette Whole-body exposure chamber Whole-body exposure chamber

11. EXPERIMENTAL DESIGN ECG tracing monitored continuously for daily changes in heart rate (HR) and heart rate variability (HRV) At 2, 4, 6 months of ETS exposure or at 3 and 6 months of CAPs exposure Ultrasound Biomicroscopy (UBM) Vascular Reactivity HistopathologyH&EImmunohistostaining

12. Ultrasound Biomicroscopy (UBM)  Vevo 660, VisualSonics, Toronto, Ontario  single-crystal mechanical transducer  central frequency: 40 MHz  focal length: 6 mm  frame rate: 30 Hz  maximum field of view of 2D imaging: 10x10 mm  spatial resolution: ~60 µm (lateral) by ~30 µm (axial)  Doppler pulse repetition frequency: up to 96 kHz, maximum unaliased velocity of 120 cm/s  Mice were anesthetized using 1.5% isoflurane  Total scan time: 30 min/mouse  Advantage: Non-invasive, Progression

13. Ultrasound Scan Procedure Anesthetized: continuous 1.5% isoflurane Total scan time: 30min/mice

14. Ultrasound Images of Aorta Plaques Ascending Aorta Aorta Arch Left Common Corotid artery Brachio- Cephalic artery Right Common Corotid artery

15. UBM Quantification Procedure 330um

16. Atherosclerosis Progression in Naïve ApoE -/- At baseline, Young: 1.5 month old; Old: 10.5 month old

17. Longevity of the ApoE -/- mice reduced by SS but not by CAPs No death occurred in ApoE -/- mice in CAPs exposed group. CAPs Study in 2003

18. SS Enhanced Atherogenesis (UBM) *

19. SS Enhanced Atherogenesis (within the same animals)

20. CAPs exposure enhanced atherogenesis in ApoE -/- mice, with accompanied increases in lipid content H&E ORO AirCAPsAirCAPs Normal Chow High Fat Chow Sun et al, JAMA, 2005

21. Comparative Atherogenic Effects of CAPs vs SS (UBM) CAPs: Aortic Arch and Bachiocephalic Artery; SS: Carotid Artery

22. Summary  UBM is a useful imaging technique to follow lesion progression in atherosclerotic mice.  Carotid artery appears to be the optimal location to evaluate atherosclerosis progression.  SS, but not CAPs, reduced longevity in ApoE -/-.  CAPs and SS exposures promote similar magnitude of change in animals susceptible to atherosclerosis, regardless of the diet.  Exacerbation of atherosclerosis is evident after 4 months of low level SS exposure or after 3 months of CAPs exposure.

23. Acknowledgements:   Qinghua Sun (Ohio State University)   Sanjay Rajagopalan (Ohio State University)   Chunli Quan   Mianhua Zhong   Ximei Jin   Qiang Li

24. Thank You !!! Contact Information: Tel: (845) 731-3560 E-mail: LCC4@nyu.eduLCC4@nyu.edu