Prof. Yong Ji, Ph.D., M.D Atherosclerosis Research Center Nanjing Medical University

1. Hydrogen sulfie donor, GYY4137, exhibits anti-atherosclerotic activity in high fat fed apolipoprotein E knockout mice Prof. Yong Ji, Ph.D., M.D Atherosclerosis Research Center Nanjing Medical University

2. Atherosclerosis These risk factors most frequently include hypertension, tobacco smoking, diabetes mellitus, obesity, and genetic predisposition; the molecular details of how they work are not yet known. Ross R. N Engl J Med. 1999.

3. Endogenously produced gases John W.etal. Antioxid. Redox Signal. 2009.

4. Cardiovascular actions of hydrogen Sulfide John W. et al. Antioxid. Redox Signal. 2009.

5. Cardiovascular actions of hydrogen Sulfide Elsey DJ. et al. Cell Biochem Funct 2010.

6. Biosynthesis of H2S H2S H2S CBS 3-MST CSE Pyridoxal-5’-phosphate L-cysteine H2S 3-mercaptopyruvate neurons astrocytes John W. et al. Antioxid. Redox Signal. 2009.

7. H2S and Atherosclerosis Role of Hydrogen Sulfide in the Development of Atherosclerotic Lesions in Apolipoprotein E Knockout Mice Wang Y, et al. Arterioscler Thromb Vasc Biol 2009.

8. H2S and Atherosclerosis ACS14 may prevent the progression of atherosclerosis by downregulating macrophage CX3CR1 expression. Zhang H, et al. Eur J Pharmacol 2012.

9. H2S and Atherosclerosis Yang G. et al. FASEB J. 2004. Yan et al. BBRC. 2006. Lagger et al. Free Radic Res. 2007 Pan LL, et al. Plos one 2011. H2S induced apoptosis of human aorta smooth muscle cells. H2S inhibited atherogenic modification of purified LDL induced by hypochlorite H2S inhibit homocysteine-induced superoxide production in rat thoracic aortic smooth muscle cells. H2S inhibit adhesion to TNF-α-activated HUVEC.

10. Controversial findings H2S regulates leukocyte trafficking in cecal ligation and puncture-induced sepsis. H2S induces proinflammatory cytokines synthesis in human monocyte cell line. Zhang H et al.J Leukoc Biol. 2007. Zhi L, et al. J Leukoc Biol. 2007.

11. Limitations Notably, considerable emphasis has also been placed on the use of NaHS as a “tool” to model the biological effects of endogenous H2S. In aqueous solution, NaHS releases large amount of H2S over a period of a few seconds. Nevertheless, this salt gives poor satisfaction for clinical uses , because the rapid release of H2S may cause adverse effects, such as acute and excessive lowering of blood pressure. Ideal H2S-donors for therapeutic purposes should generate H2S with slow releasing rates.

12. GYY4137 Characterization of a Novel, Water-Soluble Hydrogen Sulfide Releasing Molecule (GYY4137): New Insights Into the Biology of Hydrogen Sulfide GYY4137 Li L, et al. Circulation. 2008.

13. GYY4137 and NaHS GYY4137 NaHS Li L et al. Circulation. 2008.

14. Effect of hydrogen sulfide donors on LPS-induced formation of inflammatory mediators in macrophages Anti-inflammation LPS GYY4137 0-1000umol/L Macrophages Inflammation NaHS Li L et al.Free Radic Biol Med, 2009.

15. Hypothesis AS Oxide stress Inflammation Endothelial dysfunction

16. GYY4137 inhibited lipid accumulation and experssion of LOX-1 protein induced by Ox-LDL in RAW264.7 Cells A C B *P <0.05,***P < 0.001 vs CTL ; #P<0.05, ##P<0.01vs ox-LDL treated group. n=4

17. Effects of GYY4137 on H2S system in RAW264.7 Cells A B ? +P<0.05, c.f. no treatment, *P<0.05, c.f. treatment with oxLDL. n=4

18. ZYJ1122, a structural analogue of GYY4137

19. ZYJ1122, a structural analogue of GYY4137 , is unable to release H2S or affect lipid accumulation in RAW264.7 Cells B C +P<0.05, c.f. no treatment, n=4

20. ZYJ1122, a structural analogue of GYY4137 , is unable to release H2S or affect lipid accumulation in RAW264.7 Cells A B C +P<0.05, c.f. control, n=4

21. ZYJ1122, a structural analogue of GYY4137 , is unable to inhibit lipid accumulation in in RAW264.7 Cells B A C +P<0.05, c.f. no treatment, *P<0.05, c.f. treatment with ox-LDL.n=4-6

22. GYY4137, but not ZYJ1122, inhibited lipid accumulation in human peripheral blood moncyte-derived macrophages healthy donors. patient with coronary heart disease. *P <0.05, **P<0.01 vs CTL ; #P<0.05vs GYY4137.n=6-9.

23. GYY4137 inhibited chemokine releasing in ox-LDL-treated RAW 264.7 cells CXCL2 CXCR-4 CXCL10 +P<0.05, c.f. no treatment, *P<0.05, c.f. treatment with oxLDL.n=4-6

24. GYY4137 prevented ox-LDL induced macrophage iNOS expression B A +P<0.05, c.f. no treatment, *P<0.05, c.f. treatment with oxLDL.n=4

25. GYY4137 prevented oxLDL-induced macrophage NF-κB activation C A B E D *P <0.05, ,**P < 0.01, ***P < 0.001 vs no treatment ; #P<0.05, ##P<0.01 ###P<0.001vs ox-LDL treated group

26. GYY4137 prevented oxLDL-induced macrophage ICAM-1 and VCAM-1 Experssion +P<0.05, c.f. no treatment, *P<0.05, c.f. treatment with oxLDL.n=4

27. Animal experiment Eight-week-old ApoE-/- mice High-fat diet for four weeks Normal food for 30days GYY4137( 50mg/kg body weight ) or NS, (i.p. ) Oil-red O–stained aortic root lesions of mice Anesthetized and sacrificed .

28. Effects of GYY4137 on H2S system in apoE-/- mice B A ? ‡ P<0.05, c.f. WT mice, §P<0.05, c.f. ApoE-/- + NS。（n=5-8）

29. GYY4137 has no effect on body weight and plasma lipids TC:total cholesterol, TG:triglyceride, HDL-C: high-density lipoprotein-cholesterol, LDL-C: low-density lipoprotein-cholesterol. *P＜0.05, **P＜0.01, ***P＜0.001 vs WT. n=5-8 animals/group.

30. GYY4137 decreases atherosclerotic lesion size in apoE-/- mice A B **P＜0.01, ***P＜0.001 vs WT, ###P＜0.01 vs apoE-/- + NS. n=5-8 animals/group.

31. GYY4137 improved endothelium-dependent relaxation function and activated PI3K/Akt/eNOS Pathway A B D C ? *P＜0.05, ***P＜0.001 vs WT. #P＜0.05, ##P＜0.01,vs apoE-/- + NS. n=6 animals/group.

32. GYY4137 reduced superoxide formation and LOX-1 expression in aortas of apoE-/- mice A a b B **P＜0.01 vs WT mice. #P＜0.05 vs apoE-/- + NS. n=6 animals/group.

33. GYY4137 reduced TNF-α, IL-6 and ICAM-1 mRNA expression in aortas of apoE-/- mice **P＜0.01 vs WT mice. #P＜0.05 vs apoE-/- + NS. n=6 animals/group.

34. Summary