I dentification of inflammatory gene modules based on variations of human endothelial cell responses to oxidized lipids Peter S. Gargalovic, Peter S. Gargalovic, Minori Imura Imura, Bin Zhang, , Bin Zhang, Nima Nima M. M. Gharavi Gharavi, Michael J. Clark, , Michael J. Clark, Minori Joanne Pagnon Pagnon, Wen , Wen- -Pin Yang, Pin Yang, Aiqing Aiqing He, Amy Truong, He, Amy Truong, Shilpa Shilpa Joanne Patel , Stanley F. Nelson , Steve Horvath, Judith A. Berliner, Patel , Stanley F. Nelson , Steve Horvath, Judith A. Berliner, Todd G. Kirchgessner Kirchgessner, and , and Aldons Aldons J. Lusis J. Lusis Todd G.
GOAL: Understand the complex biological system/ disease Understand the complex biological system/ disease GOAL: Evolution of approaches: Evolution of approaches: 1. 1. gene cloning and single gene regulation gene cloning and single gene regulation 2. identification of gene- -gene relationships (pathways) gene relationships (pathways) 2. identification of gene 3. 3. regulation of a pathway in the given system regulation of a pathway in the given system 4. integration of a given pathway/ genome into complex 4. integration of a given pathway/ genome into complex and dynamic biological system (current challenge) and dynamic biological system (current challenge)
NEW TECHNOLOGI ES (Expression arrays): NEW TECHNOLOGI ES (Expression arrays): I nitial use in gene expression mapping: I nitial use in gene expression mapping: I dentify all genes regulated by I nflammatory Stimuli (Oxidized Lipids)
Classical approach to exploratory expression array to exploratory expression array Classical approach experiments experiments oxPAPC (4hrs) Dose response 10 μ g/ml 30 μ g/ml Data Data HAEC analysis analysis 50 μ g/ml LPS (2ng/ml) Time course Multiple time points 0 - 4hrs (50 μ g/ml) Data Data HAEC analysis analysis
Major Differences in Gene Regulation Between LPS and OxPAPC oxPAPC (50 ug/ml) Bacterial LPS (2 ng/ml) 459 70 vs. 283 17 742 genes 87 genes
Many Genes and Pathways are Regulated by Oxidized Lipids Many Genes and Pathways are Regulated by Oxidized Lipids (complex system!!!) (complex system!!!) LDL Inflammatory response Endothelial Cells Oxidation Unfolded Protein Response Nitric Oxide SREBP Oxidized Phospholipids ~ 800 genes GPCR, ERK/EGR-1 CREB/HO-1 cAMP Src/Jak/STAT
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 Identifies network modules that can be used to explain gene • regulation and function (pathway analysis) regulation and function (pathway analysis) •Hierarchical clustering with the topological overlap matrix 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
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
Genetic background influences inflammatory Genetic background influences inflammatory responses to oxidized lipids in human EC responses to oxidized lipids in human EC 1400 oxPAPC 1200 PAPC 1000 IL8 (pg/ml) 800 600 400 200 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 DONOR HAEC #
I nflammatory Responses are Preserved Between I nflammatory Responses are Preserved Between Cell Passages Cell Passages IL8 ELISA 1600 correlation=0.825 p<0.001 1400 1200 2nd (pg/ml) 1000 800 600 400 200 0 0 200 400 600 800 1000 1200 1400 1st (pg/ml)
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
Experimental Design: Experimental Design: ENDOTHELIAL CELL DONORS 1 2 3 4 5 6 7 8 9 10 11 12 TREATMENT (4hrs) 1. PAPC (40 ug/ml) 2. oxPAPC (40ug/ml) 1043 Genes Regulated by OxPAPC Data Analysis Using Gene Co-expression Network Approach
Genetic Perturbation Approach to Study Genetic Perturbation Approach to Study Gene Regulation Gene Regulation oxPAPC oxPAPC Endothelial cell line (1) Endothelial cell line (2) SREBP activity SREBP activity (+) LOW (+++) HIGH Expression of SREBP- regulated genes Expression of SREBP- regulated genes (+) LOW (+++) HIGH
1043 genes in the oxPAPC network are separated 1043 genes in the oxPAPC network are separated into 15 modules into 15 modules Topological Overlap Matrix Plot 12 cell lines
Brown Module is enriched in SREBP Pathway Genes Brown Module is enriched in SREBP Pathway Genes gene Highest INSIG1 6.257772 INSIG1 6.194221 SLC2A3 6.061201 INSIG1 5.695922 SLC2A14 5.606994 SLC2A14 5.227064 SLC2A14 4.260267 NQO1 3.984579 Brown module has SQLE 3.5742 26 genes SLC2A3 3.483622 Ranking LPIN2 3.087652 8 of 14 SREBP based on ADRB2 2.922237 connectivity SC4MOL 2.915552 targets are in Brown CYP51A1 2.373458 module CPNE8 2.241534 SQSTM1 1.861886 (p-value 1.26x10 -10 ) ) CYP51A1 1.784242 --- 1.722028 LOC285148 1.674725 --- 1.602659 --- 1.528179 SQLE 1.36481 LTB4DH 0.84509 LOC283219 0.790956 ID3 0.691711 --- 0.255479
Blue and Red module are enriched in UPR genes Blue and Red module are enriched in UPR genes RED MODULE (52 genes) BLUE MODULE (256 genes) 5 out of top 10 genes are UPR 22 out of top 100 genes are UPR genes genes CEBPB 10.82586 GIT2 9.623114 ATF4 9.178292 SLC7A5 8.612143 CEBPG 7.563844 MGC4504 7.446907 Ranking Ranking based on XBP1 7.270555 based on network KIAA0582 7.019388 network connectivity connectivity MTHFD2 6.86908 SPTLC2 6.824852 DDIT4 6.682974 EEF2K 6.475676 KIAA0582 6.40407 KIAA0121 6.288301 VEGF 6.155599 RALA 6.062034 RED module UPR BLUE module UPR LOC148418 6.031962 enrichment enrichment (p-value 1.3x10 -13 ) C14orf27 5.904039 (p-value 0.049 ) ) ) IMAP1 5.65993 MLYCD 5.586476
Gene network separates genes into modules based on Gene network separates genes into modules based on mechanism of regulation mechanism of regulation SREBP genes (Brown module) (p-value 1.26x10 -10 ) ) UPR genes (Blue and Red module) (p-value 1.3x10 -13 and 0.049 ) IL8 (Blue module) IL8 expression in cell lines is highly correlated with UPR genes IL8 expression in cell lines is highly correlated with UPR genes
Screen for UPR regulatory sites in 1043 network genes Endoplasmic Reticulum Endoplasmic Reticulum IRE1 PERK ATF6 XBP1 ATF4 UPR genes UPRE 5’-TGACGTGG-3’) XBP1 and ATF6 ERSE-I 5’- CCAAT(N9)CCACG -3’ ERSE-II 5 –ATTGGNCCACG- 3’ ATF4 C/EBP-ATF 5’-TTGCATCA -3’ CRE-like site found in IL8 promoter
ATF4 siRNA inhibits I L8 expression in primary human aortic ECs UPR UPR IL8 p=0.001 Scrambled siRNA 400 ATF4 siRNA Blue Blue mRNA (% of control) module module 300 p=0.0002 200 p=0.0006 68% 100 72% 74% 0 400 ATF4 ATF4 CONT OX TUN Scrambled siRNA ATF4 siRNA p=0.003 300 mRNA (% of control) p<0.0001 1000 SREBP SREBP INSIG1 200 Scrambled siRNA ATF4 siRNA Brown Brown 800 p<0.0001 mRNA (% of control) 100 module module 81% 600 85% 71% 0 400 CONT OX TUN 200 0 CONT OX TUN
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 400 8000 ATF4 ATF4 MGC4504 MGC4504 Scrambled siRNA Scrambled siRNA ATF4 siRNA 7000 ATF4 siRNA p=0.003 300 6000 mRNA (% of control) p<0.0001 p=0.0007 mRNA (% of control) 5000 200 4000 p=0.003 3000 p<0.0001 100 2000 p=0.0008 81% 85% 71% 1000 96% 97% 89% 0 0 CONT OX TUN CONT OX TUN
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