MR. Wisitsak phoksawat Ph.D. candidated in Biomedical Sciences Program,

1. Evidence of NKG2D/ ligands interaction promote Atherosclerosis MR. Wisitsakphoksawat Ph.D. candidated in Biomedical Sciences Program, Graduate School, KhonKaen University, Thailand. Advisor : Assoc. Prof. Dr. ChanvitLeelayuwat

2. Multi-risk factor of Atherosclerosis • Dyslipidemias • - high of low-density lipoprotein cholesterol (LDL-C) • - low of high-density lipoprotein (HDL) • Diabetes Mellitus and Impaired glucose tolerance (IGT) • Hypertension • Obesity and overweight • Cigarette smoking • Increased age, Gender, Family history and race

3. Involvement of abnormal metabolic conditions in atherosclerosis abnormal metabolic conditions ( Dyslipidemias) Stress to artherial endothelial cells (up-regulated chemokine and adhesion molecules, recruit atherogenic immune cells) Vascular inflammation Atherosclerosis (chronic inflamation and auto-immune disease)

4. Initiation of atherosclerosis Picture from Obesity, inflamation, and altherosclerosis. Nat Rav. Cardiol. (2009)

5. LDL oxidation • Nonenzymatic mechanisms • - the catalysis by transition metal ions, copper(Cu2+), iron(Fe2+), and hemin (Fe3+) • - hemoglobin (Hb) • Enzymatic mechanisms • - myeloperoxidase • - 15 or 12/15-lipoxygenase (LO) • - NADPH oxidases

6. Classification of scarvenger receptors base on structural motifs (Krieger and colleagues, 1997) • SR-AI/SR-AII, CD36, SRB1, MARCO, LOX1, SREC-1/SREC-II, SR-PSOX, Stabilin-1 recognize modified low-density lipoproteins. Picture from Role of macrophage scavenger receptors in atherosclerosis, immunobiology (2012).

7. Schematic mechanisms of scavenger receptor mediated interaction with modified lipoproteins Three steps are ; I. Recognition of the ligand II. Invagination of the membrane forming vesicles III. The degradation of modified lipoprotein ligands in lysosomes Picture from Role of macrophage scavenger receptors in atherosclerosis, immunobiology (2012).

8. T cells in altherosclerosis • Macrophage can take up, process, and present neo-antigens such as oxLDL to CD4+ T cells. • About 10 % of CD4+ T cells cloned from human lesion recognize oxLDL on MHC classII (DR). Picture from Obesity, inflamation, and altherosclerosis. Nat Rav. Cardiol. (2009)

10. Cytokines involvement in atherosclerosis Anti-athergenic cytokines Pro-athergenic cytokines TNF-α IL-1 IL-1 βIL-2 IL-12 IL-18 IFN-α IFN-ɣ TGF-β MCP-1RANTESCD40LIL-4 ? IL-6 ? TGF-β IL-5 IL-9 IL-10 IL-13 IL-33IL-1ra IL-4 ? IL-6 ? • Induce Pro-athergenicchemokinerelease • Activated adhesion molecules expression • Effects SMC migration and proliferation • Regulated immune responses promoting Th1 response • Stimulates MMP expression • Induces apoptosis • Modulates extracellular matrix expression • Induce anti-athergenicchemokinerelease • Regulated immune responses promoting Th2 response • Stimulates profibrotic state promoting wound healing and angiogenesis

11. NKG2D receptor • KLRK1 (killer cell lectin-like receptor subfamily K, member 1) • encode on human chromosome 12 within NK gene complex and chromosome 6 in mice • Type II C-type lectin-like family of transmembrane proteins • express as homodimer on NK cell,NKT cell,+ T cell and some CD8+ T cells • function both an activating and co-stimulatory receptor • bind to variety of ligands; • - Human : ULBP, RAET1, MICA,MICB • - Mouse : RAE1, H60, MULT1

12. Inhibitory and activating receptors of NK cells • inhibitory receptors • KIRs • CD94/NKG2A • ILTs • activating receptors • CD16 • NCRs • KIR2DS • CD94/NKG2C • NKG2D

14. Upregulationof NKG2D Ligands in Sera and Atherosclerotic Plaques of patients with Metabolic Dysfunction. * p<0.05 , ** p< 0.01 , *** p< 0.001 • NKG2D ligands were up-regulated in atherosclerotic plaques • Both macrophage and endothelial cells of atherosclerotic plaques expressed MICA/B

15. Preferential Upregulation of NKG2D Ligandsin Tissue and Organs of ApoE -/- Mice Where Abnormal Metabolites Accumulate • The ligands are up-regulated (10-30 fold) in the atherosclerotic aortas.

16. Preventing NKG2D/Ligand Interactions Suppresses Plaque Formation in ApoE -/-Mice * p<0.05 , ** p< 0.01 , *** p< 0.001 (reduced 5-6 fold) NKG2D plays a critical role in plaque formation in Western diet (WD)–fed and streptozotocin(STZ) -diabetic apolipoprotein E–deficient (ApoE ¯/¯) mice.

17. * p<0.05 , ** p< 0.01 , *** p< 0.001 • Oil red O staining of cross sections of aortic roots also revealed significantly reduced plaque formation in Klrk1-/-ApoE-/- mice compared with the ApoE-/- controls

18. Preventing NKG2D/Ligand Interactions Reduces the Systemic Production of Multiple Cytokine Markers Associated With Immune Activation * p<0.05 , ** p< 0.01 , *** p< 0.001 • The NKG2D/ligand-mediated immune cell activation associated with atherosclerotic progression might function at least partly through the increased production of these cytokines.

20. Acknowledgement • Associate Professor. Dr. ChanvitLeelayuwat for my advisor. • Associate Professor. Dr. NantaratKomanasin and Dr. AmornratJumniansong • Cardiovascular Research Group (CVRG), KhonKaen University, Thailand. • The Higher Education Research Promotion and National Research University Project of Thailand, Office of the Higher Education Commission, through the Center of Exellence in Specific Health Problems in Greater Mekong Sub-region Cluster (SHeP-GMS), KhonKaen University (KKU). • All members of LAB3 and CVRG groups

21. Thank you for your attention