UNIVERSITI PUTRA MALAYSIA ATHEROGENIC EVENTS...
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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