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UNIVERSITI PUTRA MALAYSIA

IN VITRO EFFECTS OF CRYPTOTANSHINONE ON EARLY ATHEROGENIC EVENTS INDUCED BY OXIDIZED LOW-DENSITY

LIPOPROTEIN AND TUMOUR NECROSIS FACTOR-α

ANG KOK PIAN

FPSK(m) 2010 16

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IN VITRO EFFECTS OF CRYPTOTANSHINONE ON EARLY ATHEROGENIC

EVENTS INDUCED BY OXIDIZED LOW-DENSITY LIPOPROTEIN AND TUMOUR

NECROSIS FACTOR-α

By

ANG KOK PIAN

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in

Fulfilment of the Requirements for the Degree of Master of Science

December 2010

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Abstract of thesis presented to the senate of Universiti Putra Malaysia in fulfilment of the

requirement for the degree of Master of Science

IN VITRO EFFECTS OF CRYPTOTANSHINONE ON EARLY ATHEROGENIC

EVENTS INDUCED BY OXIDIZED LOW-DENSITY LIPOPROTEIN AND TUMOUR

NECROSIS FACTOR-α

By

ANG KOK PIAN

December 2010

Chair: Associate Professor Dr. Zuraini Ahmad, PhD

Faculty: Faculty of Medicine and Health Sciences

Development of early atherogenic events involve endothelial cell injury by oxidized low-density

lipoprotein (oxLDL) and pro-inflammatory cytokines, such as tumour necrosis factor-α (TNF-α).

The injuries of endothelial cells lead to subsequent increase in endothelial permeability and

expression of adhesion molecules favouring monocytes’ adhesion to endothelium. In addition,

the production of nitric oxide (NO), a permeability-regulator, is also impaired in dysfunctioned

endothelium. Cryptotanshinone (CTS) is one of the major compounds isolated from the Chinese

herb Salvia milthiorrhiza, which is found to be effective against cardiovascular diseases.

However, the effects of CTS on oxLDL and TNF-α-induced early atherosclerotic events have not

been investigated. The aim of this study was to evaluate the anti-atherosclerotic effects of CTS at

pre-lesional stage by examining its effects on the endothelial permeability, expression of

adhesion molecules and chemokines, restoration of nitric oxide (NO) and adhesion of U937

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monocytic cells to human umbilical vein endothelial cells (HUVEC). OxLDL (100 μg/ml) and

TNF-α (10 ng/ml) were used to induce endothelial hyperpermeability, to increase expression of

adhesion molecules, i.e. vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion

molecules-1 (ICAM-1), adhesion of monocytes, and to suppress NO. TNF-α was also used to

induce the expression of monocyte chemoattractant protein-1 (MCP-1) in HUVEC. The results

of MTT assay showed that CTS had no cytotoxic effect to HUVEC up to 10 μM concentration.

During oxLDL-induced early atherogenic events, CTS, at 1-10 μM, significantly suppressed the

endothelial hyperpermeability and at 2.5 - 20 μM, it significantly reduced the adhesion of

monocytes to HUVEC and restored the production of NO. ICAM-1 was significantly suppressed

by 2.5 – 10 μM of CTS whereas VCAM-1 expression was suppressed by 1 – 20 μM of CTS. For

the events induced by TNF-α, 1 – 20 μM CTS significantly reduced endothelial

hyperpermeability, 1 – 10 μM CTS significantly suppressed monocytes’ adhesion to HUVEC,

the expression of ICAM-1, and at similar range of concentrations, restored NO production CTS,

at 2.5 – 10 μM, significantly suppressed the expressions of VCAM-1 and MCP-1 (P<0.05).

These findings suggest that CTS may play a role in the prevention of early or pre-lesional stage

of atherosclerosis by suppressing increased endothelial permeability and monocytes’ adhesion to

endothelium. These data indicate that the restoration of NO bioavailability may play a role in

reversing the elevated endothelial permeability, and that CTS may attenuate the recruitment of

monocytes via the suppression of adhesion molecules and chemokine’s expressions.

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi

keperluan untuk ijazah master sains

KESAN CRYPTOTANSHINONE PADA TAHAP AWAL ATEROGENIK YANG

DIARUH OLEH LIPOPROTIN TEROKSIDA KEPADATAN RENDAH DAN TUMOR

NEKROSIS FAKTOR-α IN VITRO

Oleh

ANG KOK PIAN

Disember 2010

Pengerusi: Profesor Madya Dr. Zuraini Ahmad, PhD

Fakulti: Fakulti Perubatan dan Sains Kesihatan

Perkembangan tahap awal aterogenik melibatkan kecederaan pada sel endothelia oleh lipoprotein

teroksida berdensiti rendah (oxLDL) dan pro-inflamasi sitokin, seperti tumour necrosis factor-α

(TNF-α). Kecederaan sel endotelia diikuti oleh peningkatan ketelapan endothelia dan ekspresi

molekul adhesi yang menggalakkan perlekatan sel monosit pada sel endothelia. Selain itu,

penghasilan nitrik oksida, yang merupakan satu pengawalatur ketelapan, juga terganggu pada sel

endotelia yang tidak berfungsi. Cryptotanshinone (CTS) merupakan satu sebatian utama yang

diekstrak dari Salvia milthiorrhiza yang terbukti berkesan melawan penyakit kardiovaskular.

Namun begitu, kesan CTS pada kejadian tahap awal aterogenik yang diaruh oleh oxLDL dan

TNF-α masih belum dikaji. Tujuan projek penyelidikan ini ialah untuk menilai kesan anti-

aterosklerotik CTS pada tahap awal aterosklerosis dengan mengenalpasti kesannya terhadap

ketelapan endotelia, ekspresi molekul adhesi dan kemokin, pemulihan penghasilan nitrik oxida

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(NO) dan perlekatan sel monosit U937 kepada sel endotelia vena umbilikal manusia (HUVEC).

OxLDL (100 μg/ml) dan TNF-α (10ng/ml) digunakan untuk meningkatkan ketelapan endotelia,

ekspresi molekul adhesi, i.e. molekul adhesi sel vaskular-1 (VCAM-1) dan molekul adhesi

interselular-1 (ICAM-1), adhesi sel monosit dan penurunan penghasilan NO. TNF-α juga

digunakan untuk mengaruh peningkatan ekspresi protin kemopenarikan sel monosit-1 (MCP-1)

pada HUVEC. Ujian MTT menunjukkan bahawa CTS tidak sitotoksik terhadap HUVEC

sehingga tahap kepekatan 10 μM. Semasa kejadian tahap awal aterogenik yang diaruh oleh

oxLDL, CTS, dari kepekatan 1-10 μM, menurunkan secara signifikan ketelapan tinggi endothelia,

dan dari 2.5-20 μM, ia dengan signifikannya mengurangkan perlekatan sel monosit pada

HUVEC dan mengembalikan penghasilan NO yang normal. ICAM-1 dikurangkan secara

signifikan oleh 2.5-10 μM CTS, manakala ekspresi VCAM-1 dikurangkan oleh CTS pada

kepekatan 1-20 μM. Untuk kejadian aterogenik yang diaruh oleh TNF-α, kepekatan CTS dari 1-

20 μM telah dengan signifikannya menurunkan ketelapan tinggi endotelia, CTS pada 1-10 μM

pula mengurangkan secara signifikan perlekatan sel monosit pada HUVEC, ekspresi ICAM-1

dan mengembalikan penghasilan NO kepada asal, kepekatan dari 2.5-10 μM, dengan

signifikannya menurunkan ekspresi VCAM-1 dan MCP-1 (P<0.05). Penemuan-penemuan ini

menunjukkan bahawa CTS mungkin memainkan peranan penting di dalam pencegahan kejadian

tahap awal aterogenik dengan mengurangkan kadar ketelapan tinggi endotelia dan perlekatan

monosit pada endotelia. Data ini juga menunjukkan pemulihan penghasilan NO yang mungkin

memainkan peranan untuk menurunkan kadar ketepalan tinggi endothelia, dan CTS mungkin

menghalang perlekatan sel monosit melalui pengurangan ekspresi molekul adhesi dan kemokin.

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ACKNOWLEDGEMENTS

First and foremost I offer my sincerest gratitude to my supervisor, Associate Professor Dr

Zuraini Ahmad, who has supported me throughout my research project with her patience and

knowledge whilst allowing me the room to work in my own way. I attribute the level of my

Masters degree to her encouragement and effort and without her this thesis and research project,

too, would not have been completed or written. One simply could not wish for a better or

friendlier supervisor. Above all and the most needed, she provided me unflinching

encouragement and support in various ways. Her truly scientist intuition has made her as a

constant oasis of ideas and passions in science, which exceptionally inspire and enrich my

growth as a student, a researcher and a scientist want to be. I am indebted to her more than she

knows. Many thanks go in to my project co-supervisor, Dr Abdah Md Akim for her valuable

advice in my project, using her precious time to read my manuscripts and this thesis.

I am also grateful to my laboratory mate, Mr. Felix Yong, for his constructive advice and

comment in my research project. Without him, my project would not have been completed

smoothly. Many thanks go in particular to Ms. Charlin Tham, Heng Kean, Ms. Syamimi and

Malarvili. I am much indebted to Charlin for her precious time in guiding me and help in

troubleshooting throughout the whole research progress. I have also benefited by the mental

support provided by Malarvili who always kindly grants me her time. Thanks to Heng Kean and

Syamimi for her kind assistance during my research experiment.

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I would also like to acknowledge the staffs from Physiology Laboratory and Cell Signaling

Laboratory, particularly Kak Normayati, Kak Ngah, Naz, Mr. Zul and Ayien for their kind

assistance in technical issues.

I convey my special thanks to Mr. Brian Tay, my special friend who provides me endless support

in many ways during my research stage as well as the thesis writing stage. I will never forget his

warm calls during my late nights in the laboratory rushing for experiments.

Not forgetting, I would like to express my special thanks to my parents that provided me mental

and physical supports throughout the whole process of my Master degree. Without them I would

not be able to spend 2.5 years in completing my research project and this thesis.

Finally, I would like to thank everybody who was important to the successful realization of this

thesis, as well as expressing my apology that I could not mention personally one by one.

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I certify that a Thesis Examination Committee has met on 16th

December 2010 to conduct the

final examination of Ang Kok Pian on his thesis entitled “In vitro effects of cryptotanshinone

on early atherogenic events induced by oxidized low-density lipoprotein (OXLDL) and

tumour necrosis factor – α (TNF-α)” in accordance with the Universities and University

Colleges Act 1971 and the constitution of the Universiti Putra Malaysia [P.U.(A) 106] 15 March

1998. The committee recommends that the student be awarded the degree of Master of Science

(Physiology).

Members of the Thesis Examination Committee were as follows:

Dr. Chong Pei Pei, PhD

Associate Professor

Faculty of Medicine and Health Sciences

Universiti Putra Malaysia

(Chairman)

Dr. Sabrina Sukardi, PhD

Associate Professor

Faculty of Medicine and Health Sciences

Universiti Putra Malaysia

(Internal Examiner)

Dr. Zulkhairi Hj. Amom, PhD

Associate Professor

Faculty of Medicine and Health Sciences

Universiti Putra Malaysia

(Internal Examiner)

Dr. Mahanem Mat Noor, PhD

Associate Professor

Faculty of Science and Technology

Universiti Kebangsaan Malaysia

(External Examiner)

________________________

BUJANG KIM HUAT, PhD

Professor and Deputy Dean

School of Graduate Studies

Universiti Putra Malaysia

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This thesis was submitted to the Senate of Universiti Putra Malaysia and has been accepted as

fulfillment of the requirement for the degree of Master of Science. The members of the

Supervisory Committee were as follows:

Zuraini Ahmad, PhD

Associate Professor

Faculty of Medicine and Health Sciences

University Putra Malaysia

(Chairman)

Abdah Md Akim, PhD

Faculty of Medicine and Health Sciences

University Putra Malaysia

(Member)

______________________________

HASANAH MOHD GHAZALI, PhD

Professor and Dean

School of Graduate Studies

Universiti Putra Malaysia

Date:

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DECLARATION

I declare that the thesis is my original work except for quotations and citations which have been

duly acknowledged. I also declare that it has not been previously, and is not concurrently,

submitted for any other degree at Universiti Putra Malaysia or at any other institution.

________________

ANG KOK PIAN

Date: 16 December 2010

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LIST OF FIGURES

Figure Page

2.1. The oxidative modification of LDL. 9

2.2. Leukocyte-endothelial adhesion. 20

2.3. The chemical structure of cryptotanshinone. 31

2.4. The plant of Salvia miltiorrhiza (Danshen). 32

2.5. The dried root of Salvia miltiorrhiza (Danshen). 32

3.1. Cell viability test with MTT method. 42

3.2. Effect of CTS on oxLDL-induced increased endothelial permeability. 43

3.3. Effect of CTS on oxLDL-induced adhesion of U937 monocytic cells 44

to HUVEC.

3.4. Calcein-AM tagged U937 monocytic cells binding to HUVECs 45

after treatment with various concentrations of CTS.

3.5. Effect of CTS on the oxLDL-induced sICAM-1 and sVCAM-1 47

expression in HUVECs.

3.6. Effect of CTS on oxLDL-attenuated NO production in HUVEC. 48

4.1 Effect of CTS on the TNF-α-increased endothelial permeability. 60

4.2 Effect of CTS on TNF-α-induced adhesion of U937 monocytic cells 61

to HUVEC.

4.3 Effect of CTS on the TNF-α-induced ICAM-1 and VCAM-1 63

expression in HUVECs.

4.4 Effect of CTS on the TNF-α-induced MCP-1 expression in HUVECs. 64

4.5 Effect of CTS on TNF-α-attenuated NO production in HUVECs. 65

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LIST OF ABBREVIATIONS

oxLDL Oxidized low-density lipoprotein

TNF-α Tumour necrosis factor-α

IL-1β Interleukin-1β

IFN-γ Interferon-gamma

CTS Cryptotanshinone

ICAM-1 Intercellular adhesion molecules-1

VCAM-1 Vascular cell adhesion molecules-1

MCP-1 Monocyte chemoattractant protein-1

HUVEC Human umbilical vein endothelial cell

NO Nitric oxide

LOX-1 Lectin-like oxidized LDL receptor-1

AJ Adherens junction

TJ Tight junction

GJ Gap junction

VEGF Vascular endothelial growth factor

LDL Low-density lipoprotein

JMD Juxtamembrane domain

CTD C-terminal domain

cGMP Cyclic guanosine monophosphate

eNOS Endothelial-nitric oxide synthase

iNOS Inducible-nitric oxide synthase

nNOS Neuronal-nitrix oxide synthase

JAM-C Junctional adhesion molecule-C

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M-SCF Macrophage colony stimulating factor

PECAM-1 Platelet endothelial cellular adhesion molecules

JAM-A Junctional adhesion molecule-A

IgG Immunoglobulin-G

LFA-1 Lymphocyte function-associated antigen

HMG-CoA 3-hydroxy-3-methylglutaryl-coenzyme A

DMSO Dimethyl sulfoxide

LSGS Low serum growth supplement

FBS Fetal bovine serum

RPMI Roswell Park Memorial Institute medium

PBS Phosphate buffered saline

BSA Bovine serum albumin

BCECF-AM 2’,7’-bis-(2-Carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester

MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

ELISA Enzyme-linked immunosorbent assay

ATCC American Type Culture Collection

FITC Fluorescein isothiocyanate

μM/ml Micromolar/mililitre

μg/ml Microgram/militre

μL Microlitre

ng/ml Nanogram/mililitre

ROS Reactive oxygen species

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TABLE OF CONTENTS

Page

ABSTRACT i

ABSTRAK iii

ACKNOWLEDGEMENTS v

APPROVAL SHEET 1 vii

APPROVAL SHEET 2 viii

DECLARATION ix

LIST OF FIGURES x

LIST OF ABBREVIATIONS xi

CHAPTER

1 INTRODUCTION

1.1 General Introduction 1

1.2 Research Objectives 3

2 LITERATURE REVIEW

2.1 Atherosclerosis 4

2.1.1 Pathogenesis of Atherosclerosis 5

2.2 Oxidized Low-Density Lipoprotein (OxLDL) 6

2.2.1 Oxidation of Low-Density Lipoprotein (LDL) 7

2.2.2 OxLDL and Atherosclerosis 9

2.3 Tumour Necrosis Factor–α (TNF-α) 10

2.3.1 Expression of TNF-α 11

2.3.2 Roles of TNF-α in Atherosclerosis 11

2.4 Endothelial Cell 13

2.2.1. Physiological Functions of Endothelial Cells 13

2.2.2. Endothelial Activation and Dysfunction 14

2.5 Endothelial Permeability 15

2.5.1 Mechanisms of Endothelial Permeability 16

Regulation

2.5.2 Endothelial Hyperpermeability in Atherosclerosis 18

2.6 Monocytes Recruitment 19

2.6.1 Recruitment of Monocytes in Early 21

Atherosclerosis Events

2.6.2 Differentiation of Monocytes in 22

Vascular Wall

2.7 Cellular Adhesion Molecules 22

2.7.1 Intercellular Adhesion Molecules-1 24

(ICAM-1) and Vascular Cell Adhesion

Molecules-1 (VCAM-1)

2.8 Chemokines 26

2.8.1 Monocyte Chemoattractant Protein-1 26

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(MCP-1)

2.9 Nitric Oxide (NO) 27

2.9.1 Roles of Nitric Oxide in Atherosclerosis 28

2.10 Danshen 31

2.10.1 Cryptotanshinone (CTS) 31

2.10.2 Pharmacological Effects of Cryptotanshinone 33

2.11 Simvastatin 34

3 CRYPTOTANSHINONE ATTENUATES IN VITRO

OXLDL-INDUCED PRE-LESIONAL ATHEROSCLEROSIS

EVENTS

3.1 Introduction 35

3.2 Materials and Method 37

3.2.1 Materials 38

3.2.2 Methods 38

3.2.2.1 Cell Culture 38

3.2.2.2 Cell Viability Assay 38

3.2.2.3 Measurement of Permeability of 39

HUVEC Monolayers

3.2.2.4 HUVEC-Monocyte Adhesion Assay 39

3.2.2.5 Measurement of Adhesion Molecules 40

Expression

3.2.2.6 Measurement of Nitric Oxide 40

Production

3.2.2.7 Statistical Analysis 41

3.3 Results 41

3.3.1 Effect of CTS on Cell Viability 41

3.3.2 CTS Supresses Permeability of HUVEC 42

Monolayers

3.3.3 CTS Inhibits HUVEC-Monocyte Adhesion 43

3.3.4 CTS Suppresses Adhesion Molecules 46

Expression

3.3.5 Effect of CTS on NO Level 48

3.4 Discussion 49

3.5 Conclusion 52

4 CRYPTOTANSHINONE SUPRESSES TUMOUR NECROSIS

FACTOR-α-INDUCED ENDOTHELIAL HYPERPERMEABILITY

AND MONOCYTE ADHESION IN VITRO

4.1 Introduction 53

4.2 Materials and Methods 55

4.2.1 Materials 55

4.2.2 Methods 56

4.2.2.1 Cell Culture 56

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4.2.2.2 Measurement of HUVEC Monolayers 56

Permeability by FITC-Dextran

4.2.2.3 Monocytes-Endothelial Adhesion 57

Adhesion Assay

4.2.2.4 Expression of Cellular Adhesion 57

Molecules

4.2.2.5 Expression of Chemokine- Monocyte 58

Chemoattractant Protein -1 (MCP-1)

4.2.2.6 Measurement of Nitric Oxide 58

Production

4.2.2.7 Statistical Analysis 59

4.3 Results 62

4.3.1 Effect of CTS on TNF-α-Increased HUVEC 62

Monolayer Permeability

4.3.2 Effect of CTS on TNF-α-Induced HUVEC- 64

Monocyte Adhesion

4.3.3 Effect of CTS on TNF-α-Induced Cellular 65

Adhesion Molecules Expression

4.3.4 Effect of CTS on TNF-α-Induced MCP-1 67

Expression

4.3.5 Effect of CTS on TNF-α-Attenuated NO 68

Production

4.4 Discussion 69

4.5 Conclusion 72

5 SUMMARY, CONCLUSION AND RECOMMENDATIONS 73

FOR FUTURE RESEARCHS

REFERENCES 77

APPENDICES 106

BIODATA OF STUDENT 119