Lipopolysaccharide-stimulated

responses in rat aortic

endothelial cells by a systems

biology approach

Proteomics 2006, 6, 5915–5928

Group B：林伯勳、吳益盛、紀永鑫、許梓亭、唐紹祖范文郎、白宏益

Introductionsepsis

http://juang.bst.ntu.edu.tw/

Blood organ dysfunction

LPS

LPS Cytokines

Chemokines

Intracellular mediators (NO)

TLR4

NF-κB

inflammation

Structural features of cell wall in Gram(-) bacteria

Nat Rev Drug Discov (2003)2:635

LPS, pathogen↓↓↓↓

TNF↓↓↓↓

IL-1↓↓↓↓

IL-6, IL-8, INF↓↓↓↓

NO, AA, …

Local inflammation

Monocyte/Macrophage

activation

Endothelial cell activationComplement activation

(C3a, C5a )

Systemic effects

Fever

Acute-phase

reaction

Sepsis shock

Low cardiac output

Low peripheral resistance

DICARDS

DIC: disseminated intravascular coagulationARDS : adult respiratory distress syndrome

Introduction

Tissue damage

Cytotoxic effect

Redox-mediated inflammatory responses

plenty of DATA

System biology

humaninflammatory

Other model×

Sepsis

Inflammation

Oxidative stress

Global study

Bioinformatics ：BioCarta, KEGG, Gene Ontology database

Introduction

LP

S

cDNA microarray

2-DE MALDI-TOF MS/MS

Cytokine array

Rat aortic endothelial cell

Endothelial cells

PI (propidium iodide)

vWF (von Willebrand factor)

Purity: > 95%

Cascade of LPS-induced activation of endothelial cells and leukocytes

Cardiovasc Res (2003) 60:49

LPS Stimulation

Flow chart

BGSSJ software

to classify and annotate

by molecular function based on GO databasespecific genes

integrated biologicalpathways

TranscriptomicsTranscriptomicsTranscriptomicsTranscriptomics assay assay assay assay

(cDNA microarray))))

Functional classification of signaling

pathway by BioCarta

Cholesterol efflux pump

cytochrome P450 family

metabolic activation or inactivation

of environmental toxins or xenobiotics,

in inflammation and infection mechanisms

alterations

in drug clearance

and toxin activation

during inflammatory

responses

Functional classification of

metabolic pathway by KEGG

Fructose-1,6-bisphosphatases

anabolism and catabolis

m

gluconeogenesis drugs

Expression changes of proteins by LPS-treated

in rat Ekes by MALDI-Q-TOF MS/MS

Antioxidative

H2O2 ↓ -Cys-SO3H ＞ -Cys-SO2H

Acidic isoform increase

endothelial toward infiltration by leukocytes

cell contractility, migration, stiffness, stiffening,

proliferation, and wound healing

modulate iron homeostasis

In inflammation plays a cytoprotective and

antioxidant function to sequestrate iron

Expression changes of proteins by LPS-treated

in rat Ekes by MALDI-Q-TOF MS/MS

Proteasome inhibitors strongly stabilize the

protein IkBa to selectively inhibit the activation

of NF-kB in the inflammatory response.

elimination cytotoxic

dismutation superoxide radical

to O2 and H2O2

SODM → primary antioxidant defense system × H2O2

Peroxiredoxin 1 → eliminating H2O2

Dysregulation of the redox cellularimbalance is an important phenomenon for Inflammatory response.

peptide biosynthesis and protein degradation

anti-inflammatory drug

In lysosome vesicles

Secreted proteins assay from

LPS-activated rat ECsControl

LPS 24h

• TIMP-1

• VEGF

• Cytokine: IL-6

• Chemokines:

– C chemokines :

– CC chemokines : MCP-1, MIP-3αααα.

– CXC chemokines : CINC-2, -3, and LIX.

– CX3C chemokines : Fractalkine

BGSSJ annotation of transcriptomics &

proteomics data

Cellular Component

Molecular Function

Biological Process

Venn diagram of “response to

stimulus” annotated by BGSSJ

Venn diagram of “immune

response” annotated by BGSSJ

Genes:Il1b, Ccl21b, Fcgr3, Aif1, Ahsg, and Fgg)

Proteins:

LIX, MIP-3αααα, MCP-1, CINC-2, and CINC-3)

A possible NF-κκκκB associated responses and

self-protected responses in LPS-stimulated ECs

ProliferationAtherogenesisInflammation

Apoptosis

Anti-oxidation

Anti-inflammation

Anti-apoptosis

LPS → human coronary artery ECsLDL pathwayMSP/RON receptor signaling pathway

could inhibit the transcriptional activity

Summary• Through a proteomic study, we can provide more

accurate and correct biomarkers in inflammation studies.– PSA6, cathepsin B, IL-6, and chemokines may play a

promotional role in inflammatory responses. – Peroxiredoxin 1, SODM, FRIH, and FRIL1, as an anti-

inflammatory or self-protective function.

• These biomarkers and proposed pathways might enable us to develop new diagnosis markers for inflammatory diseases.

• More importantly, these biomarkers may serve as new drug targets (proteasome inhibitor and cathepsin B inhibitor) for septic therapeutic studies in the future.

• By systems biology analysis can integrate many powerful viewpoints to help explain complicated biological phenotypes or diseases which haven’t been explored in previous studies.