Novel function of NADPH oxidase in atherosclerosis. Yun Soo Bae Department of Life Science Ewha Womans University

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1 Novel function of NADPH oxidase in atherosclerosis Yun Soo Bae Department of Life Science Ewha Womans University

2 Recent understanding of ROS: act as second messengers e e Catalase/peroxidase O 2 H 2 O 2 H 2 O SOD e OH. (mild) (mild) e O 2.- (toxic) Cytokines Growth factors GPCR ligands UV and X-ray Pathophysiology Mitochondria NADPH oxidases High Low Quiescence Proliferation _. O H 2 O 2 2 Apoptosis Necrosis

3 GPCR Integrin RTK TNFR AC DD PKA G a bg NF-kB JNK PLCb PKC. O 2 H 2 O 2 STATs MAPK PLCg PTK Smads PTK PI3K S/T kinase S/T kinase TGFbR Apoptosis Jak TCR Cytokine R BCR ROS production: temporal (5-10 min) and spatial production Second messenger: Scavenging of ROS by addition of antioxidants blocks agonist-dependent cell signaling It occurs in plasma membrane, not mitochondria: NADPH oxidase

4 Transmembrane topology of NADPH oixdase(nox) isozymes: Connection of Nox with various receptors Lambeth JD, Nature Rev. Immunol. 4, 181, 2004

5 What is atherosclerosis? Atherosclerosis involves the formation in the arteries of lesions that are characterized by inflammation, lipid accumulation, cell death, and fibrosis. Hansson and Libby Nature Rev. Immunol 6; 508 (2006) Inflammatory mechanisms couple dyslipidaemia to atheroma formation. Libby Nature 420; 868 (2002) Concerted action of lipid accumulation and inflammation Inflammation processes in MØ and EC play an important role in initiation and early progression of atherosclerosis

6 Toll-like receptor signalling. Receptor Ligand Origin of ligand TLR1 Triacyl lipopeptides Bacteria and mycobacteria TLR2 TLR3 Lipoprotein/lipopep tides Peptidoglycan Lipoteichoic acid Double-stranded RNA Various pathogens Gram-positive bacteria Viruses TLR4 Lipopolysaccharide Gram-negative bacteria TLR5 Flagellin Bacteria TLR6 TLR7 TLR8 TLR9 Diacyl lipopeptides Lipoteichoic acid Single-stranded RNA Single-stranded RNA CpG-containing DNA Mycoplasma Gram-positive bacteria Viruses Viruses Bacteria and viruses TLR10 not determined not determined TLR11 not determined Uropathogenic bacteria Gene expression Cytokines Adhesion molecules chemokines Type I IFN Gene expression MyD88-dependent pathway MyD88-independent pathway NATURE REVIEWS 2004

7 TLR4-induced proinflammatory activation in human coronary artery cells Reduced atherosclerosis in MyD88-null mice links elevated serum cholesterol levels to activation of innate immunity signaling pathways. Bjorkbacka H et al. Nature Med. 10: (2004) Lack of Toll-like receptor 4 or myeloid differentiation factor 88 reduces atherosclerosis and alters plaque phenotype in mice deficient in apolipoprotein E. Michelsen KS et al. Proc Natl Acad Sci U S A. 101: (2004).

8 Production of ROS and ICAM-1/MCP-1 play an important role in early event of atherogenesis TLR4 LPS NOX4 TLR4 MCP-1 IL-8 N C ICAM-1 H 2 O 2 MCP-1 IL-8 JBC 276; (2001) J. Immunol. 173; 3589 (2004) Cardiovas Res 72; 447 (2006)

9 What is high risk endogenous molecule causing atherosclerosis? Extenstive oxidation TLR4/MD2/CD14 TLR2/CD36 Pro-inflammatory cytokines Triggers early events of atherosclerosis Miller et al., JBC 2003; Miller et al., MBC 2003; Miller et al., ATVB 2005; Boullier et al., ATVB 2006

10 mmldl and oxldl induce generation of ROS but not nldl in J774 cells Cell count A Control mmldl 5 μg/ml mmldl 10 μg/ml mmldl 25 μg/ml mmldl 50 μg/ml B Control nldl mmldl OxLDL DCF fluorescence DCF fluorescence

11 Activation of gp91phox (Nox2) by assembly of regulatory proteins in phagocytes Lambeth JD, Nature Rev. Immunol. 4, 181, 2004

12 Nox2-defective macrophage fails to ROS generation in response to mmldl Relative fluorescence (%) J774 cells mmldl Relative fluorescence (%) Peritoneal macrophage mmldl Control virus Nox2 virus Wild type Nox2 KO Control virus Nox2 virus Nox2 GAPDH

13 Signaling network by mmldl in macrophage mmldl TLR4/MD2/CD14 activation Syk activation PLCγ-p PKC Nox2 H 2 O 2 Pro-inflammatory cytokines production (RANTES, IL-6) Migration of smooth muscle cell

14 Signaling pathways of mmldl/lps for macrophage activation TLR4 MD2 CD14 mmldl Indirect activation MD2 CD14 LPS Direct activation Nox2 Nox4 TLR4 MØ Syk PKC ROS EC PLCγ Circ Res 104; 210 (2009) Circ Res 104; 1355 (2009) NFkB J. Immunol. 173; 3589 (2004) Cardiovas Res 72; 447 (2006) Augmented expression of pro-inflammatory cytokines and stimulated the migration of SMC

15 Toll-like receptor signalling. Receptor Ligand Origin of ligand TLR1 Triacyl lipopeptides Bacteria and mycobacteria TLR2 TLR3 Lipoprotein/lipopep tides Peptidoglycan Lipoteichoic acid Double-stranded RNA Various pathogens Gram-positive bacteria Viruses TLR4 Lipopolysaccharide Gram-negative bacteria TLR5 Flagellin Bacteria TLR6 TLR7 TLR8 TLR9 Diacyl lipopeptides Lipoteichoic acid Single-stranded RNA Single-stranded RNA CpG-containing DNA Mycoplasma Gram-positive bacteria Viruses Viruses Bacteria and viruses TLR10 not determined not determined TLR11 not determined Uropathogenic bacteria Gene expression Cytokines Adhesion molecules chemokines Type I IFN Gene expression MyD88-dependent pathway MyD88-independent pathway NATURE REVIEWS 2004

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17 Interaction between TLR2 and ROS generation system A B TLR2 interacts with Nox1, but not with Nox4

18 Functional analysis of the interaction between TLR2 and Nox1 A B Aortic SMC Nox1 is required for Pam3CSK4-induced ROS generation in ASMCs

19 Nox1-dependent SMC migration and restenosis in ligation injury model A WT Nox1K/O N=5

20 Pam3CSK4-induced of cytokines in murine ASMC A: Real time PCR B: ELISA for MIP-2

21 Proinflammation events in atherogenesis Cell 104: (2001)

22 Pam3CSK4-dependent SMC stimulates U937 monocyte adhesion to HAEC A Relative adhesion cells(%) B Relative adhesion cells(%) P < 0.01 * MIP-2(pg/ml) * P < 0.05 ** P < 0.05 ** (-)pam3csk4 (+)pam3csk4 SMC w/ or w/o Pam3CSK Culture supernatant containing MIP2 Adhesion analysis with HAEC and U937 in Transwell 0 WT sup Nox1 KO sup

23 A Pam3CSK4-dependent SMC stimulates U937 transendothelial migration Relative migration cells(%) P < * MIP-2(pg/ml) P < * P < ** SMC w/ or w/o Pam3CSK Culture supernatant containing MIP2 B Relative migration cells(%) P value < 0.01 (-)pam3csk4 (+)pam3csk4 Transmigration analysis with HAEC and U937 in Transwell 0 WT Nox1 KO

24 Conclusions Pam3CSK4 Membrane TLR2 TLR1 Nox1 SMC Cytoplasm C N H 2 O 2 Cell migration Nox1 plays an important role in TLR2-mediated ROS generation which in turn stimulates SMC migration and vascular remodeling.

25 Cell migration assay in EC Can Nox isozymes be good drug targets in restenosis or atherosclerosis?

26 Suppressive effects of Nox inhibitor on balloon-injured neointima formation Control Nox inhibitor

27 Immunohistochemistry analysis Control VEGFRII SMA VEGFRII SMA Merge Merge

28 Development of Nox inhibitor 120 e - HTS Readout Lucigenin (red) Lucigenin (ox) detect Inhibition rate (%) 화합물 #1 화합물 #2 화합물 #3 화합물 #4 화합물 #5 화합물 #6 10uM 2.5uM 0.625uM 0.156uM 0.039uM 0.01uM e - e - Control In vivo Control In vitro LPS LPS + hit#5 inhibitor Cell migration assay ChemGenomics 25,000 Lead

29 Acknowledgments Department of Life Sciences Ewha Womans University Jee Hyun Lee: mmldl/tlr4 Jee Hyun Lee, Sunah Kim: Pam3CSKTLR2 Hye Sun Park: LPS/TLR4 Department of Medicine UCSD Dr. Joseph Witztum Dr. Yury Miller Professor Sang Won Kang