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Peter S. Gargalovic,

Identification of inflammatory gene modules based on variations of human endothelial cell responses to oxidized lipids. Peter S. Gargalovic,

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Peter S. Gargalovic,

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  1. Identification of inflammatory gene modules based on variations of human endothelial cell responses to oxidized lipids Peter S. Gargalovic, Minori Imura, Bin Zhang, Nima M. Gharavi, Michael J. Clark, Joanne Pagnon, Wen-Pin Yang, Aiqing He, Amy Truong, Shilpa Patel , Stanley F. Nelson , Steve Horvath, Judith A. Berliner, Todd G. Kirchgessner, and Aldons J. Lusis

  2. GOAL: Understand the complex biological system/disease • Evolution of approaches: • gene cloning and single gene regulation • identification of gene-gene relationships (pathways) • regulation of a pathway in the given system • integration of a given pathway/genome into complex and dynamic biological system (current challenge)

  3. NEW TECHNOLOGIES (Expression arrays): Initial use in gene expression mapping: Identify all genes regulated by Inflammatory Stimuli (Oxidized Lipids)

  4. Classical approach to exploratory expression array experiments oxPAPC (4hrs) Dose response 10 μg/ml 30 μg/ml Data analysis HAEC 50 μg/ml LPS(2ng/ml) Time course Multiple time points 0 - 4hrs (50 μg/ml) Data analysis HAEC

  5. 459 70 283 17 Major Differences in Gene Regulation Between LPS and OxPAPC oxPAPC (50 ug/ml) Bacterial LPS (2 ng/ml) vs. 742 genes 87 genes

  6. Unfolded Protein Response SREBP Nitric Oxide ERK/EGR-1 GPCR, cAMP Src/Jak/STAT CREB/HO-1 Many Genes and Pathways are Regulated by Oxidized Lipids (complex system!!!) LDL Inflammatory response Oxidation Endothelial Cells Oxidized Phospholipids ~ 800 genes

  7. Can we take advantage of the large amount of data collected from differentially perturbed states to learn more about the biological system? • Approach: Weighted Gene Co-expression NETWORK Analysis (WGCNA) • Identifies network modules that can be used to explain gene regulation and function (pathway analysis) • Hierarchical clustering with the topological overlap matrix • Uses intramodular connectivity to identify important genes • References • Bin Zhang and Steve Horvath (2005) "A General Framework for Weighted Gene Co-Expression Network Analysis", Statistical Applications in Genetics and Molecular Biology: Vol. 4: No. 1, Article 17. • Horvath S, Zhang B, Carlson M, Lu KV, Zhu S, Felciano RM, Laurance MF, Zhao W, Shu, Q, Lee Y, Scheck AC, Liau LM, Wu H, Geschwind DH, Febbo PG, Kornblum HI, Cloughesy TF, Nelson SF, Mischel PS (2006) "Analysis of Oncogenic Signaling Networks in Glioblastoma Identifies ASPM as a Novel Molecular Target", PNAS

  8. Hypothesis: Genetic variation modulates inflammatory responses to oxidized phospholipids in human population • Interleukin 8: • Pro-inflammatory cytokine implicated in atherogenesis • Mediates adhesion of monocytes to EC • Highly induced by oxPAPC • IL8 levels are higher in patients with unstable CAD then in healthy individuals • Elevated plasma IL8 levels are associated with increased risk for future CAD

  9. Genetic background influences inflammatory responses to oxidized lipids in human EC

  10. Inflammatory Responses are Preserved Between Cell Passages

  11. Co-Expression Network of Endothelial Responses to Oxidized Phospholipids ENDOTHELIAL CELL DONORS 1 2 3 4 5 6 7 8 9 10 11 12 Oxidized Phospholipids EXPRESSION PATTERNS IL8 Gene X Gene Y

  12. Experimental Design: ENDOTHELIAL CELL DONORS 1 2 3 4 5 6 7 8 9 10 11 12 TREATMENT (4hrs) • PAPC (40 ug/ml) • oxPAPC (40ug/ml) 1043 Genes Regulated by OxPAPC Data Analysis Using Gene Co-expression Network Approach

  13. Genetic Perturbation Approach to Study Gene Regulation Expression of SREBP- regulated genes (+)LOW Expression of SREBP- regulated genes (+++) HIGH oxPAPC oxPAPC Endothelial cell line (1) Endothelial cell line (2) SREBP activity (+) LOW SREBP activity (+++) HIGH

  14. 1043 genes in the oxPAPC network are separated into 15 modules Topological Overlap Matrix Plot 12 cell lines

  15. Brown Module is enriched in SREBP Pathway Genes gene Highest Brown module has 26 genes 8 of 14 SREBP targets are in Brown module (p-value 1.26x10-10) Ranking based on connectivity

  16. CEBPB 10.82586 GIT2 9.623114 ATF4 9.178292 SLC7A5 8.612143 CEBPG 7.563844 MGC4504 7.446907 Ranking based on network connectivity XBP1 7.270555 KIAA0582 7.019388 MTHFD2 6.86908 SPTLC2 6.824852 DDIT4 6.682974 EEF2K 6.475676 KIAA0582 6.40407 KIAA0121 6.288301 VEGF 6.155599 RALA 6.062034 LOC148418 6.031962 C14orf27 5.904039 IMAP1 5.65993 MLYCD 5.586476 Blue and Red module are enriched in UPR genes RED MODULE (52 genes) 5 out of top 10 genes are UPR genes BLUE MODULE (256 genes) 22 out of top 100 genes are UPR genes Ranking based on network connectivity RED module UPR enrichment (p-value 0.049 ) BLUE module UPR enrichment (p-value 1.3x10-13 )

  17. Gene network separates genes into modules based on mechanism of regulation IL8 (Blue module) IL8 expression in cell lines is highly correlated with UPR genes SREBP genes (Brown module) (p-value 1.26x10-10 ) UPR genes (Blue and Red module) (p-value 1.3x10-13 and 0.049)

  18. UPRE 5’-TGACGTGG-3’) ERSE-I 5’- CCAAT(N9)CCACG -3’ ERSE-II 5 –ATTGGNCCACG- 3’ C/EBP-ATF 5’-TTGCATCA -3’ XBP1 and ATF6 ATF4 CRE-like site found in IL8 promoter Screen for UPR regulatory sites in 1043 network genes Endoplasmic Reticulum IRE1 PERK ATF6 XBP1 ATF4 UPR genes

  19. ATF4 siRNA inhibits IL8 expression in primary human aortic ECs UPR Blue module IL8 ATF4 SREBP Brown module INSIG1

  20. Co-expression network can be applied to new gene-function discovery (MGC4504 in red module is regulated by ATF4) Gene of unknown function present in UPR module ATF4 MGC4504

  21. SUMMARY • Common genetic variations in human population have significant impact on inflammatory responses to oxidized lipids • Genetic variation-based gene co-expression network approach was used to: • subdivide genes into pathways based on mechanism of regulation (UPR versus SREBP pathway) • predict UPR involvement in regulation of IL8 and MGC4504 • ER homeostasis and associated stress pathways may play a central role in mediating endothelial inflammatory responsiveness to oxidized phospholipids and possibly other stimuli

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