S1P (Edg) receptors in macrophages & atherosclerosis

1. S1P (Edg) receptors in macrophages & atherosclerosis Lynn Hedrick, PhD Stephen Rich, PhD Center for Public Health Genomics Robert M. Berne Cardiovascular Research Center University of Virginia

2. Early events in atherosclerosis Li & Glass, 2002

3. Sphingomyelin Ceramide Sphingosine Sphingosine-1-Phosphate Synthesis of sphingosine-1-phosphate (S1P) Sphingomyelin Deacylase Sphingomyelin Synthase Sphingomyelinase Sphingosylphosphorylcholine Ceramide Synthase Ceramidase LysoPLD Activity Sphingosine Kinase I & II Lipid Phosphatases (LPPs & SPPs) P-ethanolamine + hexadecanal lyase

4. Huwiler, Andrea and Josef Pfeilschiefter. Altering the Sphingosine-1-Phosphate/Ceramide Balance: A Promising Approach for Tumor Therapy. Current Pharmaceutical Design. 2006: (4625-4635).

5. S1P • S1P is released from mast cells, platelets, and monocytes in circulation. • S1P in plasma is found on HDL, and may contribute to the observed anti-inflammatory effects of HDL on vascular endothelium. • Plasma levels of S1P are in the nanomolar range: ~200-500nM

6. Functions of S1P in vessel wall • vasculogenesis • endothelial migration • endothelial permeability • T-lymphocyte homing • DC differentiation/maturation • inflammation role--importance in resolving inflammation

7. Sphingosine Kinases • 2 isoforms, SphK1 and Sphk2 • Regulate the balance of the lipid mediators ceramide, sphingosine & S1P in the cell. • Activated by cytokines, such as TNF. • Specific regulation is complex and not fully understood. • SphK1 and SphK2 have somewhat redundant functions in cells, but may be regulated differently. Alemany et al. Regulation and functioal roles of sphingosine kinases. NSAP. 2007:413-428.

8. S1P receptors Keul et al. ATVB. 27, 607 (2007) ‘FTY720 reduces atherosclerosis in ApoE deficient mice’ Sphingosine-1-phosphate Atherosclerosis Nofer et al. Circulation. 115, 501 (2007) ‘FTY720 inhibits the development of atherosclerosis in LDLR deficient mice’ agonist S1P1 (edg1) S1P2 (edg5) S1P3 (edg3) S1P4 (edg6) S1P5 (edg8) Activation of many downstream signaling events in vascular cells through Gi, Gq, and G12/13 signaling.

9. Extracellular actions S1P Apoptotic cell clearance ABCA1 ABCG1 Cholesterol Efflux S1P Intracellular actions SK LPP SPH Pro-survival Anti-inflammatory cytokines S1P in macrophages S1P1 Gi βγ S1P2 Gq βγ

10. Macrophage polarization M-S1P-MF “alternative activation” M2-MF / type II-MF “alternative activation” M1-MF / type I-MF “classical activation” Inducers: INFg + LPS or TNF IL10 S1P IL4 or IL13 M-S1P M1 M2a M2c Mediators: • IL10, • CCL18 • MR, SRA, SRB • TGFbVEGF • IL1, IL6, IL12, IL23, TNF, INFg • CCL2, CCL3, CCL5 • CCR7, TLR2, TLR4 • MHC II • iNOS • ROI, RNI • IL10 • Arginase 1 • TGF • IL10 • CCL17,CCL22 • CD163, MR, SRA, • SRB, Decoy-IL1RII • Arginase-1 • Polyamines Immunoregulation, Matrix deposition, Tissue remodeling Th1 responses: Type I inflammation, Bacterial killing, Tumor resistance Resolving inflammation? Th2 responses: Type II inflammation, Allergy, parasital killing Function: Modified after Mantovani, 2004, Trends in Immunology

11. ‘M-S1P’ phenotype…  = increased = decreased - = undetectable ? = unknown

12. Edg1 (S1P1)

13. Edg5 (S1P2)

14. MESA • For Edg1, 10 haplotype-tagging SNPs identified and 6 genotyped & informative. --early analysis shows some associations with inflammatory markers in Caucasian, Hispanic, and Asian populations, but not with African-American population. • For Edg5, 7 haplotype-tagging SNPs identified and 6 genotyped & informative. --early analysis shows very little association with inflammatory markers.

15. Edg1 (S1P1) & Inflammation Caucasians • No associations with Edg5 (S1P2) and any inflammatory markers phenotyped thus far, confirming mouse data. • Need TNF, IL8, IL10, TGF, IL12 phenotypes measured in MESA to accurately assess macrophage polarization.

16. Associations of Edg1 w/ T2D in MESA Caucasians • No association of Edg5 SNPs with T2D. • 2 SNPs were also associated with T2D in African-American population.

17. Summary • Edg1 and Edg5 are novel candidate genes for inflammation & atherosclerosis. • Significant associations were observed for Edg1 SNPs and markers of inflammation and T2D risk. • No significant associations were observed for Edg5 SNPs and these phenotypes. • Preliminary analyses suggest no strong associations of Edg1 and Edg5 SNPs with lipid phenotypes. • Additional phenotyping in MESA for markers of inflammation and macrophage subsets will be extremely useful.

18. Future Directions • Additional analyses including • Haplotypes • Ancestry Informative Markers (AIMs) • Multivariable modeling • G x G (joint effects of Edg1 and ABCG1) • Permutation testing • MESA Ancillary study to explore the genotype-phenotype relationships in macrophages. • Continue to explore Edg1/Edg5 in atherosclerosis using knockout mouse models.