RAR - جامعة الكوفة - كلية الصيدلة

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A STUDY OF THE EFFECTS OF SILDENAFIL AND
VARDENAFIL ON AORTIC INTIMA - MEDIA
THICKNESS AND BLOOD FLOW VELOCITIES OF
AORTA, RENAL ARTERY AND INTRA RENAL
ARTERY IN HYPERLIPIDEMIC MALE RABBITS
A THESIS
Submitted To The College Of Medicine And The Committee Of
Postgraduate Studies Of Kufa University In Partial Fulfillment
Of The Requirements For The Degree Of Master Of Science In
Pharmacology And Therapeutics
BY
Karrar Hussein Kammona
B.Sc. Pharmacy
Supervised by
Dr. Najah R. AL-Mousawi
Professor of Clinical
Pharmacology and Therapeutics
Ph.D,FRCP,FACP
1430 A.H
Dr. Akeel AM. H. Zwain
Lecturer of
Cardiovascular Physiology
Ph.D U.K.
2009 A.D.
We certify that this thesis was prepared under our supervision at the College
of Medicine, University of Kufa , as a partial requirement for the Degree of
Master of science in Pharmacology and Therapeutics.
Signature …………..
Signature……………
Name: Najah R. AL-Mousawi
Name: Akeel AM. H. Zwain
Professor of Clinical
Pharmacology and Therapeutics
Ph.D, FRCP, FACP
Supervisor
Lecturer of
Cardiovascular Physiology
Ph.D U.K.
Supervisor
In view of the available recommendations . I put forward this thesis for
debate by the examining committee .
Signature……………
Name: Najah R. AL-Mousawi
Professor of Clinical
Pharmacology and Therapeutics
Head Department of
Pharmacology and Therapeutics
Acknowledgement
Praise be to our Almighty Allah, the Gracious who gives me the power and
motivation to perform and present this work.
I wish to express my heartfelt gratitude and appreciation to my honorable
supervisor Prof. Dr. Najah R. Al Mousawi for his guidance, valuable
advices and support.
I wish to address special thanks to Dr. Akeel AM.H. Zwain, my honorable
supervisor for his great help and support in the field of Doppler ultrasound
examinations and measurements .
I am deeply grateful to Kufa College of Medicine, represented by the Dean
and the staff for providing facilities required for this work.
I deeply appreciate Dr. Hussein Abed Al-khahdam for his kind help.
I am so thankful to Assistant prof. Dr. Majed K. Hussein & Dr Ahmed
Al-Mohana for their kind help.
Also I would like to express my deepest thanks to Dr. Liwaa H. Mahdi AlKulabi who help me in the field of histopathology.
I highly appreciate Assistant prof. Dr Abdul-Kareem Al-Mayah for his
help in statistical analysis.
My gratitude and appreciation to pharmacist Muhammad Ali & Hayder
Chassep for their kind helps.
I am grateful to Mr. Fares Abbas for his help in blood sampling.
Finally, my heartfelt appreciation and gratitude to My Family for their
infinite support and patience during the time of the study.
Karar H. Kammona
List of Contents
Subject
Page
Dedication
І
Acknowledgments
ІІ
List of Contents
ІІІ
List of tables
IX
List of figures
XІІ
List of abbreviations
XVІ
Summary
XIX
Chapter One: Introduction and Literature Review
1.1 Atherosclerosis
1
1.1.1 Mortility and morbidity
1
1.1.2 Risk factor of atherosclerosis
2
1.1.3 Pathogenesis of atherosclerosis
2
1.1.3.1 Hypotheses of atherogenesis
2
1.2 Oxidative Stress
6
1.2.1 Free radicals
6
1.2.2 Oxidative stress and reactive oxygen species
7
1.2.3 Sources and reactions of ROS and RNS
7
1.2.4 Antioxidants
8
1.2.5 Oxidative stress in atherosclerosis
9
1.3 Endothelium dysfunction
10
1.3.1 Endothelial dysfunction as an initial step of atherosclerosis
10
1.3.2 Oxidative stress and endothelial dysfunction
11
1.3.3 Antioxidant properties of nitric oxide in atherosclerosis
12
1.4 Treatment of Atherosclerosis
13
1.4.1 Lifestyle change
13
1.4.2 Medications
13
1.4.3 Therapeutic approach
14
1.5 Phosphodiesterase Inhibitors.
15
1.5.1 Sildenafil (Viagra)
15
1.5.1.1 Mechanism of action and pharmacological properties
15
1.5.1.2 Effect on hemodynamics
16
1.5.1.3 Pharmacokinetics and metabolism
17
1.5.1.4 Adverse effects
18
1.5.1.5 Drug interaction
18
1.5.2 Vardenafil (Levitra®)
18
1.5.2.1 Mechanism of action and pharmacological properties
19
1.5.2.2 Hemodynamic effects
19
1.5.2.3 Pharmacokinetics
20
1.5.2.4 Side effects
21
1.5.2.5 Drug interaction
21
1.6 Doppler ultrasound technique
21
1.6.1 Doppler parameters and Doppler indices
22
1.7 Blood flow rate and blood flow velocity
24
1.7.1 Definition of blood flow and blood flow velocity
24
1.7.2 Factors affecting on blood flow and blood flow velocity
24
1.7.3 Effect of atherosclerosis on blood flow velocity
25
1.7.4 Aortic blood flow
26
1.7.5 Renal artery blood flow
26
1.7.6 Intra-renal arteries blood flow
27
1.8 Aim of the study
28
Chapter Two: Material and Methods
2.1 Materials
29
2.2 Animals and study design
30
2.3 Drugs
32
2.3.1 Sildenafil (viagra)
32
2.3.2 Vardenafil (levatra)
32
2.4 Preparation of sample
32
2.4.1 Blood sampling
32
2.4.2 Tissue sampling
32
2.5 Measurement of samples
34
2.5.1 Measurement of serum total cholesterol
34
2.5.2 Measurement of Triglycerides Concentration
35
2.5.3 Measurement of HDL-C Concentration
37
2.5.4 Calculation of LDL,VLDL and Atherogenic index
38
2.5.5 Measurement of serum malondialdehyde (MDA) Level
38
2.5.6 Determination of serum reduced glutathione (GSH)
39
2.6 Measurement of aortic diameter, intima-media thickness and
blood flow velocities of the aorta, renal artery and intra-renal
arteries
42
2.7 Calculation of Doppler indices
43
2.8 Histopathological procedure
43
2.9 Statistical analysis
44
Chapter Three: Results
3.1 Effect on serum total cholesterol (TC) level
45
3.2 Effect on serum triglyceride (TG) level
46
3.3 Effect on HDL-C level
48
3.4 Effect on serum LDL-C level
49
3.5 Effect on VLDL-C level
51
3.6 Effect on atherogenic index
52
3.7 Effect on serum level of Malon Dialdehyde MDA .
54
3.8 Effect on serum reduced glutathione level GSH .
55
3.9 Effect on aortic intima-media thickness (IMT)
57
3.10 Effect on aortic diameter
58
3.11 Effect on aortic peak systolic blood flow velocity (PSV)
60
3.12 Effect on aortic end diastolic blood flow velocity (EDV)
61
3.13 Effect on aortic blood flow resistive index (RI)
63
3.14 Effect on aortic blood flow pulsatality index (PI)
64
3.15 Effect on renal artery peak systolic blood flow velocity(PSV)
66
3.16 Effect on renal artery end diastolic blood flow velocity(EDV)
67
3.17 Effect on renal artery blood flow resistive index (RI)
69
3.18 Effect on renal artery blood flow pulsatality index (PI)
70
3.19 Effect on intra-renal arteries peak systolic blood flow velocity
72
3.20 Effect on intra-renal arteries end diastolic blood flow velocity
73
3.21 Effect on intra-renal arteries blood flow resistive index (RI)
75
3.22 Effect on intra-renal arteries blood flow pulsatality index (PI)
76
3.23 Effect on renal artery-to-aortic peak systolic velocity ratio
78
3.24 Histopathological findings
79
Chapter Four: Discussion
4.1 Effect on Serum Lipid Profile
89
4.1.1 Effect of cholesterol enriched diet on serum lipid profile
89
4.1.2 Effect of Sildenafil and Vardenafil treatment on serum lipid
profile
89
4.2 Effect on oxidative stress
89
4.2.1 Effect of high cholesterol diet on oxidative stress
89
4.2.2 Effect of Sildenafil and Vardenafil on MDA and GSH level
90
4.3 Effect on aortic intima-media thickness
91
4.3.1 Effect of cholesterol enriched diet on aortic intima-media
thickness
91
4.3.2 Effect of Sildenafil and Vardenafil treatment on aortic intimamedia thickness
91
4.4 Effect on aortic diameter
92
4.4.1 Effect of cholesterol enriched diet on aortic diameter
92
4.4.2 Effect of Sildenafil and Vardenafil treatment on aortic
diameter
93
4.5 Effect on peak systolic blood flow velocity
93
4.5.1 Effect of cholesterol enriched diet on peak systolic velocity
93
4.5.2 Effect of Sildenafil and Vardenafil treatment on peak systolic
velocity
94
4.6 Effect on End diastolic blood flow velocity
95
4.6.1 Effect of cholesterol enriched diet on end diastolic velocity
95
4.6.2 Effect of Sildenafil and Vardenafil treatment on end diastolic
velocity
96
4.7 Effect on resistive index (RI)
96
4.7.1 Effect of cholesterol enriched diet on resistive index
96
4.7.2 Effect of Sildenafil and Vardenafil treatment on resistive
index
97
4.8 Effect on pulsatality index (PI)
98
4.8.1 Effect of cholesterol enriched diet on pulsatality index
98
4.8.2 Effect of Sildenafil and Vardenafil treatment on pulsatality
index
98
4.9 Effect on renal artery-to-aortic peak systolic velocity ratio
99
4.9.1 Effect of cholesterol enriched diet on renal artery –to-aortic
peak systolic velocity ratio (RAR)
99
4.9.2 Effect of Sildenafil and Vardenafil treatment on renal artery –
to-aortic peak systolic velocity ratio (RAR)
99
4.10 Effect on aortic histological change
100
4.10.1 Effect of cholesterol enriched diet on aortic histology
100
4.10.2 Effect of Sildenafil and Vardenafil on aortic histology
100
Chapter five : Conclusions and Recommendations
5.1 Conclusions
101
5.2 Conclusions Recommendations
102
References
References
103
List of Tables
Table
Page
Table (1): list of chemicals, pharmaceuticals, reagent and their
supplier
29
Table (2): Instrument and their supplier
30
Table (3): Changes of serum TC level in mg/dl of the four
experimental groups
45
Table(4): Multiple comparisons between different groups mean
values of serum TC level in (mg/dl).
Table(5): Changes of serum TG level in mg/dl of the four
experimental groups.
45
Table(6): Multiple comparisons between different groups mean
values of serum TG level in ( mg/dl)
47
Table (7): Changes of serum HDL-C level in mg/dl of the four
48
47
experimental groups.
Table (8) : Multiple comparisons between different groups mean
values of serum HDL-C level in ( mg/dl) .
Table (9): Changes of serum LDL-C level in mg/dl of the four
experimental groups.
Table (10) : Multiple comparisons between different groups mean
values of serum LDL-C level in ( mg/dl) .
Table (11): Changes of serum VLDL-C level in mg/dl of the four
experimental groups.
Table (12) : Multiple comparisons between different groups mean
values of VLDL-C level .
Table(13): Changes of atherogenic index
experimental groups.
in of the four
48
50
50
51
51
53
Table (14) : Multiple comparisons between different groups mean
values of atherogenic index .
53
Table (15): Sequential changes of serum MDA level mmole/L of
the four experimental group.
54
Table (16): Multiple comparisons among different groups mean
values of serum MDA level mg/dl .
54
Table (17): Sequential changes of serum GSH level uM of the four
experimental groups.
56
Table (18): Multiple comparisons among different
groups mean values of serum GSH level .
56
Table (19): Effect of cholesterol enriched diet , Vardenafil and
Sildenafil treatment on the rabbits aortic intima-media
thickness .
57
Table (20) : Multiple comparisons between different groups mean
values of rabbits aortic intima-media thickness .
57
Table (21): Effect of cholesterol enriched diet , vardenafil and
sildenafil treatment on rabbits aortic diameter .
59
Table (22): Multiple comparisons between different groups mean
values of rabbits aortic diameter .
59
Table (23): Effect of cholesterol enriched diet , vardenafil and
sildenafil treatment on the rabbits aortic peak systolic
blood flow velocity .
Table (24): Multiple comparisons between different groups mean
values of rabbits aortic peak systolic blood flow velocity.
Table (25): Effect of cholesterol enriched diet , vardenafil and
sildenafil treatment on the rabbits aortic end diastolic
blood flow velocity .
Table (26): Multiple comparisons between different groups mean
values of rabbits aortic end diastolic blood flow velocity .
60
60
62
62
Table (27): Effect of cholesterol enriched diet , vardenafil and
sildenafil treatment on the rabbits aortic blood flow
resistive index .
63
Table (28): Multiple comparisons between different groups mean
values of rabbits aortic blood flow resistive index .
63
Table (29): Effect of cholesterol enriched diet , , vardenafil and
sildenafil treatment on the rabbits aortic blood flow
pulsatality index .
65
Table (30): Multiple comparisons between different groups mean
values of rabbits aortic blood flow pulsatality index .
65
Table (31): Effect of cholesterol enriched diet , vardenafil and
sildenafil treatment on the rabbits renal artery peak
systolic velocity.
66
Table (32): Multiple comparisons between different groups mean
values of rabbits renal artery peak systolic blood flow
velocity .
66
Table (33): Effect of cholesterol enriched diet , , vardenafil and
sildenafil treatment on the rabbits renal artery end
diastolic velocity .
68
Table (34): Multiple comparisons between different groups mean
values of rabbits renal artery end diastolic blood flow
velocity .
Table (35): Effect of cholesterol enriched diet , , vardenafil and
sildenafil treatment on the rabbits renal artery blood flow
resistive index .
68
69
Table (36): Multiple comparisons between different groups mean
values of rabbits renal artery blood flow resistive index .
69
Table (37): Effect of cholesterol enriched diet , , vardenafil and
sildenafil treatment on the rabbits renal artery blood flow
pulsatality index .
71
Table (38): Multiple comparisons between different groups mean
values of rabbits renal artery blood flow pulsatality
index .
71
Table (39): Effect of cholesterol enriched diet , , vardenafil and
sildenafil treatment on the rabbits intra- renal arteries
peak systolic velocity .
72
Table (40): Multiple comparisons between different groups mean
values of the rabbits intra- renal arteries peak systolic
velocity .
72
Table (41): Effect of cholesterol enriched diet , vardenafil and
sildenafil treatment on the rabbits intra- renal arteries end
diastolic velocity .
74
Table (42): Multiple comparisons between different groups mean
values of rabbits intra- renal arteries end diastolic
velocity .
74
Table (43): Effect of cholesterol enriched diet , vardenafil and
sildenafil treatment on the rabbits intra-renal arteries
blood flow resistive index .
75
Table (44): Multiple comparisons between different groups mean
values of rabbits intra-renal arteries blood flow resistive
index .
75
Table (45): Effect of cholesterol enriched diet , , vardenafil and
sildenafil treatment on the rabbits intra-renal arteries
blood flow pulsatality index .
77
Table (46): Multiple comparisons between different groups mean
values of rabbits intra-renal arteries blood flow
pulsatality index .
77
Table (47): Effect of cholesterol enriched diet , , vardenafil and
sildenafil treatment on rabbits renal artery-to-aortic peak
systolic velocity ratio .
78
Table (48): Multiple comparisons between different groups mean
values of rabbits renal artery-to-aortic peak systolic
velocity ratio .
78
Table (49): Demonstration of different aortic atherosclerotic phases
for different rabbit group at the end of 12 weeks of the
study
80
Table (50): Demonstration of different aortic atherosclerotic lesions
for different rabbit group at the end of 12 weeks of the
study
81
List of Figures
Figure
Page
Figure (1): Effect of Vardenafil and Sildenafil on serum TC level
in comparison to the two control groups (normal and
induced untreated).
46
Figure(2): Effect of vardenafil and sildenafil treatment on serum
TG level in comparison to the two control groups (
normal and induced untreated.
47
Figure (3): Effect of sildenafil 5mg/kg/day and verdenafil
18mg./kg/day treatment on serum HDL-C level in
comparison to the two control group (normal and
induced untreated).
49
Figure (4): Effect of vardenafil and sildenafil treatment on serum
LDL-C level in comparison to the two control groups
(normal and induced untreated)
50
Figure (5): Effect of vardenafil and sildenafil treatment on serum
VLDL-C level in comparison to the two control groups
(normal and induced untreated)
52
Figure (6): Effect of vardenafil and sildenafil treatment on
atherogenic index in comparison to the two control
group ( normal and induced untreated)
53
Figure (7) : The change in MDA Level in the four experimental
group
55
Figure (8): The change in Serum GSH Level in the four
experimental groups
56
Figure (9): Effect of Vardenafil and Sildenafil treatment on rabbits
aortic intima-media thickness (IMT) measured in (mm)
in comparisons to the two control group (normal and
58
induced untreated)
Figure (10): Effect of vardenafil and sildenafil treatment on rabbits
aortic diameter measured in (mm) in comparisons to
the two control group (normal and induced untreated)
59
Figure (11): Effect of vardenafil and sildenafil treatment on rabbits
aortic peak systolic velocity (PSV) measured in (cm/s)
in comparisons to the two control group ( normal and
induced untreated)
61
Figure (12): Effect of vardenafil and sildenafil treatment on rabbits
aortic end diastolic velocity (EDV) measured in (cm/s)
in comparisons to the two control group ( normal and
induced untreated)
62
Figure (13): Effect of vardenafil and sildenafil treatment on rabbits
aortic blood flow resistive index (RI) in comparisons to
the two control group (normal and induced untreated)
64
Figure (14): Effect of vardenafil and sildenafil treatment on rabbits
aortic blood flow pulsatality index (PI) in comparisons
to the two control group (normal and induced
untreated)
65
Figure (15): Effect of vardenafil and sildenafil treatment on rabbits
renal artery peak systolic velocity (PSV) measured in
cm/s in comparisons to the two control group (normal
and induced untreated)
67
Figure (16): Effect of vardenafil and sildenafil treatment on rabbits
renal artery end diastolic velocity (EDV) measured in
cm/s in comparisons to the two control group (normal
and induced untreated)
68
Figure (17): Effect of vardenafil and sildenafil treatment on rabbits
renal artery blood flow resistive index (RI) in
comparisons to the two control group (normal and
70
induced untreated)
Figure (18) : Effect of vardenafil and sildenafil treatment on rabbits
renal artery blood flow pulsatality index (PI) in
comparisons to the two control group (normal and
induced untreated)
71
Figure (19): Effect of vardenafil and sildenafil treatment on the
rabbits intra- renal arteries peak systolic velocity (PSV)
measured in cm/s in comparisons to the two control
group (normal and induced untreated)
73
Figure (20): Effect of vardenafil and sildenafil treatment on rabbits
intra- renal arteries end diastolic velocity (EDV)
measured in cm/s in comparisons to the two control
group (normal and induced untreated)
74
Figure (21): Effect of vardenafil and sildenafil treatment on rabbits
intra-renal arteries blood flow resistive index (RI) in
comparisons to the two control group (normal and
induced untreated)
76
Figure (22): Effect of vardenafil and sildenafil treatment on rabbits
intra-renal arteries blood flow pulsatality index (PI) in
comparisons to the two control group (normal and
induced untreated)
77
Figure (23): Effect of vardenafil and sildenafil treatment on rabbits
renal artery-to-aortic peak systolic velocity ratio in
comparisons to the two control group (normal and
induced untreated)
79
Figure (24): Percentages of aortic involvement with different
atherosclerotic lesion phases
81
Figure (25): Percentages of aortic involvement with different
atherosclerotic lesions.
82
Figure (26): A cross section of normal rabbit aorta shows the normal
appearance of arterial wall
82
Figure (27): A cross section of hypercholesterolemic rabbit's aorta
demonstrating appearance of lipid laden macrophages
(Foam cells) .
83
Figure (28): A cross section of hypercholesterolemic rabbit's aorta
demonstrating many lipid laden macrophages (Foam
cells) .
83
Figure (29): A cross section from aorta shows a pathological
intimal thickening with a poorly formed fibrous cap.
84
Figure (30): A cross section from aorta shows a pathological
intimal thickening with a poorly formed fibrous cap.
The section stained with haematoxylin and eosin (
×10)
84
Figure (31): A cross section of hypercholesterolemic rabbit's aorta
demonstrating endothelial cell with underlying Foam
cells . Intial lesion (phase 2 atherosclerosis). The
section stained with haematoxylin and eosin ( ×40)
85
Figure (32): A cross section from aorta shows a fibro-atheromatous
plaque with a thin layer of fibrous connective tissue
overlying a largely necrotic, fatty mass. Advance
atherosclerotic lesion (phase V). The section stained
with haematoxylin and eosin ( ×10)
85
Figure (33): A B-mode ultrasound image showing a rabbit
abdominal aorta with normal aortic wall thickness , D1
is the intima-media thickness = 0.3 mm , D2 is the
aortic diameter = 2.1 mm
86
Figure (34): A B-mode ultrasound image showing a rabbit
abdominal aorta with an increased aortic thickness
(sclerotic wall), but there is no apparent plaques , D1 is
the intima-media thickness = 0.6 mm , D2 is the aortic
diameter = 2.8 mm
86
Figure (35):AB-mode ultrasound image showing a rabbit abdominal
aorta with an increased aortic thickness (sclerotic wall),
there also dissemination of plaques inside the aorta ,
D1 is the intima media thickness = 0.7 mm
87
Figure(36): A Doppler wave form spectrum showing a rabbits
aortic blood flow velocity, PSV=0.49 m/s , EDV=0.06
m/s RI=0.88 , S/D ratio=8.2
87
Figure (37): A Doppler wave form spectrum showing a rabbits
renal artery blood flow velocity, PSV=0.48 m/s ,
EDV=0.17m/s, RI=0.58 , S/D ratio=2.4
88
Figure(38): Doppler wave form spectrum showing a rabbits intrarenal artery blood flow velocity, PSV=0.49 m/s ,
EDV=0.20m/s, RI=0.59 , S/D ratio=2.5
88
List of Abbreviations
Abbreviations
Meaning
ACE inhibitor
Angiotonsine converting enzyme inhibnitor
Ach
Acetylcholine
ANOVA
Analysis of ovariance
BP
Blood pressure
BT
Bleeding time
CAD
Coronary Artery Disease
cAMP
Cyclic adenosine mono phosphate
cGMP
Cyclic Guanosine Monophosphate
CRP
C-Reactive protein
DTNB
5,5-dithiobis (2-nitrobenzoic acid)
e NOs
Endothelial nitric oxide synthase
ED
Erectile dysfunction
EDCFs
Endothelium-derived contracting factors
EDRFs
Endothelium-derived relaxing factors
EDTA
Ethylenediamine tetra-acetic acid
EDV
End diastolic velocity
ET-1
Endotheline-1
FDA
Food and Drug Administration
GPx
Glutathione peroxidase
GSH
Glutathione
GssG
Oxidized Glutathione
GTP
Guanine triphosphate
HDL
High density lipoprotein
HF
Heart failure
HMG-CO-A
3-Hydroxy-3-methylglutaryl coenzyme A
HR
Heart rate
IC50
Half-maximal inhibition
ICAM
Inter cellular adhesion molecules
LDL
Low density lipoprotein
LPL
Lipoprotein lipase
MDA
Malondialdehyde
MMPs
Matrix metalloproteinases
MV
Mean velocity
NADPH
Nicotinamide Adenine Dinucleotide Phosphate
NAION
Non-arteritic ischaemic optic neuropathy
NO
Nitric Oxide
oxLDL
Oxidized LDL
PAP
Pulmonary artery pressure
PDEIs
Phosphodiesterase enzyme inhibitors
PDEs
Phosphodiesterase enzymes
PH
Pulmonary hypertension
PI
Pulsatality index
PMNs
Polymorphonuclear leukocytes
PPAR
Peroxisome proliferator activated receptor
PSV
Peak systolic velocity
RAR
Renal artery-to Aortic peak systolic velocity Ratio
RAS
Renal artery stenosis
RI
Resistive index
RNS
Reactive nitrogen species
ROS
Reactive Oxygen species
RVSP
Right ventricular systolic pressure
S/D ratio
Systolic velocity/ Diastolic velocity ratio
SEM
Standard error of mean
SMCs
Smooth muscle cells
SOD
Superoxide dismutase
TAB
Thiobarbituric
TC
Total cholesterol
TCA
Trichloroacetic acid
TG
Triglyceride
VCAM
Vascular cellular adhesion molecules
VLDL
Very low density lipoprotein
XO
Xanthine oxidase
Summary
Atherosclerosis is the major world wide killing disease. The most
common risk factors are hyperlipidemia, diabetes, arterial hypertension,
cigarette smoking and obesity. PDEIs (sildenafil and vardenafil) are the main
drugs used in the treatment of erectile dysfunction, and promise in treatment
of atherosclerosis.
Method : Twenty four local domestic male rabbits were involved in this
study. The animals were randomly divided in to four groups , Group I
rabbits fed normal chow (oxiod) diet for 12 weeks. Group II rabbits fed 2%
cholesterol enriched diet for 12 weeks. Group III rabbits fed with
cholesterol enriched diet for 6 weeks, and then continued on cholesterol
enriched diet and treated with sildenafil 5mg./kg./day orally for the next 6
weeks. Group IV rabbits fed with cholesterol enriched diet for 6 weeks ,and
then continued on cholesterol enriched diet and treated with vardenafil
18mg./kg./day orally for the next 6 weeks.The blood samples were collected
at the start of the study, 6weeks of the study and then every week through
the 6 weeks of treatment course. Serum lipids profile [Total cholesterol
(TC), Triglyceride (TG), High density lipoprotein (HDL), Low density
lipoprotein (LDL),Very low density lipoprotein
(VLDL)] ,atherogenic
index and oxidative stress parameters [GSH,MDA] were measured. Aortic
diameter and intima- media thickness and aortic, renal artery and intra-renal
artery blood flow velocities [ Peak systolic velocity (PSV), End diastolic
velocity(EDV), Pulsatality index (PI), Resistive index (RI), and Renal
artery -to- Aortic peak systolic velocity Ratio (RAR)] were measured by
triplex Doppler machine at the start of the study, 6weeks, and then at the end
of the study. Autopsy of aortic sectioning for histopathology was done at the
end of the study.
Results: 2%cholesterol enriched diet results in significant increase (P<0.05)
in serum level of TC, TG, HDL, LDL, VLDL, atherogenic index with
decrease serum GSH level and significant increase (P<0.05) in serum MDA
level. Also there was asignificant increase (P<0.05) in aortic diameter ,
intima-media thickness, PI, RI, renal artery PSV, PI, RI, intra-renal artery
PSV, PI, and RI in comparison to the normal control group. Also there was
significant increase (P<0.05) in Renal artery -to- Aortic peak systolic
velocity Ratio (RAR).
There was no significant change (P>0.05) in aortic PSV and EDV,
and no significant change (P>0.05) in renal artery and intra-renal artery
EDV.
In regard to histopathological results, all rabbits fed cholesterol enriched
diet show development of different phases of atherosclerosis and there was
significant difference (P<0.05) between the induced untreated group and the
normal control group.
Treatment of rabbits with sildenafil and vardenafil results in non
significant reduction (P<0.05) in serum level of TC, TG,HDL, LDL,
VLDL, and atherogenic index, significant reduction (P<0.05) in MDA level
and significant increase (P<0.05) in serum GSH level. There was non
significant reduction (P<0.05) in aortic diameter and intima-media thickness.
Also there was significant reduction (P<0.05) in renal artery and intra-renal
artery PSV, EDV, PI and RI. There was no significant change ( p>0.05) in
aortic PSV, EDV, PI , RI and in Renal artery -to- Aortic peak systolic
velocity Ratio (RAR) in comparison to the rabbits in the induced untreated
control group. Regarding histopathological results, sildenafil and vardenafil
treated rabbits showed atherolytic effect, but this effect did not reach the
significant level (P>0.05) in comparison to the induced untreated group.
There was no significant difference (P>0.05) between sildenafil treated
group and vardenafil treated group in measured parameters.
Conclusions: Sildenafil and vardenafil do not affect serum level of TC, TG,
LDL, VLDL, HDL and atherogenic index, but they increase serum MDA
level and decrease serum GSH level. Also both drugs insignificantly reduce
severity of atherosclerosis and reduce intima- media thickness of arteries in
treated animals. Therefore, both drugs diminish the arterial stiffness as
indicated by the reduction of PI and RI of abdominal aorta, and reduction
of PSV, EDV, PI and RI of renal artery and intra-renal arteries of the
treated animals.
1.1 Atherosclerosis
Atherosclerosis is a disease of large and medium-sized muscular
arteries and is characterized by endothelial dysfunction, vascular
inflammation, and the buildup of lipids, cholesterol, calcium, and cellular
debris within the intima of the vessel wall. This buildup results in plaque
formation, vascular remodeling, acute and chronic luminal obstruction,
abnormalities of blood flow, and diminished oxygen supply to target
organs(1).
1.1.1 Mortility and morbidity
Arteriosclerosis of the coronary and peripheral vasculature is the
leading cause of death among men and women in the United States and
worldwide(2).Estimates for 2005 in the United States alone are that about 16
million people have atherosclerotic heart disease and 5.8 million have
stroke. Cardiovascular disease, primarily coronary and cerebrovascular
atherosclerosis, caused almost 870,000 deaths in 2005—almost twice as
many as cancer caused and 9 times as many as injuries caused(3).
The incidence of all forms of atherosclerosis increases with age even
among the elderly. However, during the last 30 years, the mortality rate for
atherosclerosis has markedly and progressively decreased in the USA and in
several other industrialized countries. The decreasing mortality rate is
probably primarily due to the widespread use of preventive strategies,
resulting in decreased prevalence of such risk factors as untreated
hypertension, abnormal lipid levels, and cigarette smoking. Improvements in
diagnosis and treatment of established atherosclerosis probably have
contributed less(4).
1.1.2 Risk factor of atherosclerosis
A number of risk factors are associated with cardiovascular disease
and these may be classified into two categories: fixed and modifiable. Fixed
risk factors include genetic composition, age, menopausal status, and gender.
The modifiable factors are a series of environmental cues and lifestyle
choices including (but not limited to) diet, smoking, status of concurrent
diseases (e.g. diabetes), exercise and ethanol consumption. When
considering these risk factors it is noteworthy that many, if not all,
contribute to disease progression at least in part via oxidative stress (5).
1.1.3 Pathogenesis of atherosclerosis
1.1.3.1 Hypotheses of atherogenesis
Over the past 150 years, there have been numerous efforts to explain
the complex events associated with the development of atherosclerosis. In
this endeavor, three distinct hypotheses have emerged that are currently
under active investigation. These hypotheses of atherosclerosis are not
mutually exclusive but rather emphasize different concepts as the necessary
and sufficient events to support the development of atherosclerotic lesions.
For the purposes of this review, we refer to these paradigms as follows: 1)
the response-to-injury, 2) the response-to-retention, and 3) oxidative
modification (6). In response-to-injury hypothesis, the proposed initial step in
atherogenesis is endothelial denudation leading to a number of
compensatory responses that alter the normal vascular homeostatic
properties. For example, injury enhances endothelial adhesiveness for
leukocytes and platelets and alters the local vascular anticoagulant milieu to
a procoagulant one. Recruited leukocytes and platelets then release
cytokines, vasoactive agents, and growth factors that promote an
inflammatory response that is characterized by migration of smooth muscle
cells into the intima and their proliferation to form an intermediate lesion(7).
Another inflammatory response is the recruitment of macrophages
into the arterial wall which take up deposited LDL lipid to form lipid-laden
"foam cells," the hallmark of an early atherosclerotic lesion. The process of
lipid accumulation and foam cell formation perpetuates an inflammatory
response that perpetuates macrophage and lymphocyte recruitment (8,9).
Continued inflammation allows for cellular necrosis with a concomitant
release of cytokines, growth factors, and proteolytic enzymes that set the
stage for autocatalytic expansion of the lesion to form a space-occupying
collection in the intima not unlike an abscess that would form in other
tissues. As the lesion enlarges, it begins to encroach upon the lumen and,
ultimately, blood flow is impaired(10).
This response-to-injury hypothesis was originally based on the notion
of endothelium desquamation as a principal event initiating atherosclerosis
(10,11)
. More recently, it has become clear that endothelial desquamation is
not common and that an intact endothelial cell layer covers developing
atherosclerotic lesions. These facts, among others, promoted refinement of
the initial hypothesis such that endothelial dysfunction is sufficient to initiate
atherogenesis through increased endothelial permeability to atherogenic
lipoproteins(12).
2. The response-to-retention hypothesis
This hypothesis submits that the lipoprotein retention is the inciting
event for atherosclerosis. Within 2 h of injecting LDL into rabbits, arterial
retention of LDL and its microaggregates can be observed (13). The
underlying mechanisms involved in this process are just now coming to
light. It is estimated that 85% of subendothelial lipoprotein delivery is the
result of transcytosis, and this process is restricted to particles <70 nm in
diameter(14). This size restriction is important as it suggests that lipoprotein
lipase activity is needed for triacylgycerol-rich lipoproteins to reach the
subendothelial space(15). The retention of lipoproteins within the arterial
wall, however, appears tightly linked to components of the extracellular
matrix. Apolipoprotein, the single protein associated with LDL, is retained
within the arterial wall in close association with arterial proteoglycans (16).
This interaction is mediated by specific residues that (17), when mutated,
protect experimental animals against the development of atherosclerosis(17,18).
In addition to proteoglycan binding, lipolytic and lysosomal enzymes
in the extracellular matrix also appear to play a role. For example,
lipoprotein lipase enhances the adherence of LDL in vitro (19), and this effect
is independent of enzymatic activity(20). Once retained within the arterial
wall, LDL can form microaggregates(13,21), as well as lysosomal enzymes
such as cathepsin D and lysosomal acid lipase(22). Most importantly,
aggregated LDL is avidly taken up by macrophages and smooth muscle
cells(23) and thus can support foam cell formation(24). Thus many features of
atherosclerosis can be attributed to enhanced retention of LDL within the
arterial wall and its association with proteoglycans(6).
3. The oxidative modification hypothesis
The oxidative modification hypothesis focuses on the concept that
LDL in its native state is not atherogenic. However, LDL modified
chemically is readily internalized by macrophages through a so-called
"scavenger receptor" pathway(25). Exposure to vascular cells in medium that
contains transition metals also results in modification of LDL such that it
serves as a ligand for the scavenger receptor pathway(26). It is now clear that
one mechanism whereby cells in vitro render LDL a substrate for the
scavenger receptor pathway is via oxidation of LDL lipids and the resulting
modification of apolipoprotein (27). These observations form the basis for the
oxidative modification hypothesis of atherosclerosis , in which LDL
traverses the subendothelial space of lesion-prone arterial sites. During this
process, LDL lipids are subject to oxidation and, as a consequence,
apolipoprotein lysine groups are modified so that the net negative charge of
the lipoprotein particle increases(28). This modification of apolipoprotein
renders LDL susceptible to macrophage uptake via a number of scavenger
receptor pathways producing cholesterol ester-laden foam cells(29). It is this
accumulation of foam cells that forms the nidus of a developing
atherosclerotic lesion.
The process of LDL oxidation is associated with a number of other
potentially proatherogenic events. For example, during the initial stages of in
vitro LDL oxidation, modification of LDL lipids can occur in the absence of
any changes to apolipoprotein. Such modified LDL has been termed
"minimally modified LDL" and shown in vitro to induce the synthesis of
monocyte chemotactic protein-1 in both smooth muscle and endothelial
cells(30,31), resulting in the recruitment of inflammatory cells (32). This
particular step appears critical as mice lacking the receptor for monocyte
chemotactic protein-1 are resistant to atherosclerosis(33,34). More heavily in
vitro oxidized LDL, commonly termed "ox-LDL," is chemotactic for
monocytes(35) and T lymphocytes(36), perhaps as the result of
lysophosphatidylcholine formed during oxidation(27). Oxidized LDL has also
been shown to stimulate the proliferation of smooth muscle cells(37) and to be
immunogenic by eliciting the production of autoantibodies (38,39) and the
formation of immune complexes that can also facilitate macrophage
internalization of LDL(40,41). The recruitment of inflammatory cells may
result in the continued oxidation of LDL, setting the stage for catalytic
expansion of the atherosclerotic lesion and the full-blown spectrum of
atherosclerosis.
In summary, the hypotheses of atherosclerosis have each attempted to
explain the complex cellular events of atherosclerosis along a common
theme. The response-to-injury hypothesis focuses on vascular injury as the
inciting event in atherosclerosis with the entire spectrum of the disease
representing an attempt to heal an ongoing vascular insult. In contrast, the
response-to-retention hypothesis uses lipoprotein-matrix interactions as the
critical event in early atherosclerosis, whereas the oxidative modification
hypothesis requires oxidation of LDL lipids. Although each hypothesis
points to its own critical initiating event, there are many common features
among these competing hypotheses. For example, each involves a significant
component of inflammation, a known feature of atherosclerosis . Each
hypothesis also includes LDL as a central element, an important point as
reduction in LDL cholesterol is among the most effective means of treating
atherosclerosis. Perhaps the most unique, however, is the oxidative
modification hypothesis as it alone proposes a particular importance of
oxidative events and redox reactions in the genesis of vascular disease (6).
1.2 Oxidative Stress
Oxidative stress which can be defined as an imbalance develops
between the production of ROS and the efficacy of the cell's antioxidant
defense, leading to an altered redox status which can contribute to
endothelial dysfunction and/or cell death(42,43).
1.2.1 Free radicals
Free radicals can be defined as molecules or molecular fragments
containing one or more unpaired electrons. The presence of unpaired
electrons usually confers a considerable degree of reactivity upon a free
radical. Those radicals, derived from oxygen, represent the most important
class of such species generated in living systems (44). These free radicals are
highly reactive, unstable molecules react with (oxidize) various cellular
components including DNA, proteins, lipids / fatty acids and advanced
glycation end products (e.g. carbonyls) which lead to DNA damage,
mitochondrial malfunction, cell membrane damage and eventually cell death
(apoptosis - which is the term for programmed cell death)(45).
Free radicals are generally reactive oxygen species (ROS) or reactive
nitrogen species(RNS), and have consequences for cells as a result of one of
three factors: (i) an increase in oxidant generation, (ii) a decrease in antioxidant protection, or (iii) a failure to repair oxidative damage (46). Examples
of free radicals (oxidizing molecules) are hydrogen peroxide, hydroxyl
radical, nitric oxide, peroxynitrite, single oxygen, superoxide anion and
peroxyl radical(45).
1.2.2 Oxidative stress and reactive oxygen species
In the vascular system, the formation of ROS from endothelial cells,
smooth muscle cells (SMCs) and macrophages seems to be of major
relevance in atherogenesis in part due to their reaction with nitric oxide
(NO). NO, perhaps the most important endothelium-derived vasorelaxing
factor, is scavenged by ROS. NO reacts with ROS to yield peroxynitrite,
which can rearrange to form nitrate and the highly reactive OH radical and
the last is toxic to tissues and cells. Important pathophysiological
consequences of enhanced ROS formation in the vascular system are: (i)
attenuation of endothelium-dependent dilation, resulting in disturbed organ
perfusion and systemic hypertension, (ii) induction of cellular damage and
inflammation, (iii) induction of apoptosis, and (iv) initiation of several
intracellular signaling processes (47).
1.2.3 Sources and reactions of ROS and RNS
ROS can be produced from both endogenous and exogenous
substances. Potential endogenous sources include mitochondria, cytochrome
P450 metabolism, peroxisomes, and inflammatory cell activation (48).
Mitochondria have long been known to generate significant quantities of
hydrogen peroxide. The hydrogen peroxide molecule does not contain an
unpaired electron and thus is not a radical species. Under physiological
conditions, the production of hydrogen peroxide is estimated to account for
about ∼2% of the total oxygen uptake by the organism. After the
determination of the ratios of the mitochondrial generation of superoxide to
that of hydrogen peroxide, the former was considered as the stoichiometric
precursor for the latter. Ubisemiquinone has been proposed as the main
reductant of oxygen in mitochondrial membranes(48). Mitochondria generate
approximately 2–3 nmol of superoxide/min per mg of protein, the ubiquitous
presence of which indicates that it is the most important physiological source
of this radical in living organisms(48). Since mitochondria are the major site
of free radical generation, they are highly enriched with antioxidants
including GSH and enzymes, such as superoxide dismutase (SOD) and
glutathione peroxidase (GPx), which are present on both sides of their
membranes in order to minimise oxidative stress in the organelle(49).
Superoxide radicals formed on both sides of mitochondrial inner membranes
are efficiently detoxified initially to hydrogen peroxide and then to water by
Cu, Zn-SOD (SOD1, localised in the intermembrane space) and Mn- SOD
(SOD2, localised in the matrix). Besides mitochondria, there are other
cellular sources of superoxide radical, for example xanthine oxidase (XO), a
highly versatile enzyme that is widely distributed among species (from
bacteria to man) and within the various tissues of mammals(50). Xanthine
oxidase is an important source of oxygen-free radicals. It is a member of a
group of enzymes known as molybdenum iron–sulphur flavin hydroxylases
and catalyzes the hydroxylation of purines. In particular, XO catalyzes the
reaction of hypoxanthine to xanthine and xanthine to uric acid. In both steps,
molecular oxygen is reduced, forming the superoxide anion in the first step
and hydrogen peroxide in the second(44). Additional endogenous sources of
cellular reactive oxygen species are neutrophils, eosinophils and
macrophages. Activated macrophages initiate an increase in oxygen uptake
that gives rise to a variety of reactive oxygen species, including superoxide
anion, nitric oxide and hydrogen peroxide(51). Cytochrome P450 has also
been proposed as a source of reactive oxygen species, through the induction
of cytochrome(44) .
1.2.4 Antioxidants
Under normal conditions, numerous cellular antioxidant systems exist
to defend against oxidant stress and maintain the redox balance of the cell.
ROS are cleared from the cell by enzymatic systems including superoxide
dismutases (SODs), catalase, and glutathione peroxidase, or the
nonenzymatic system including alpha-tocopherol (vitamin E), ascorbic acid
(vitamin C), glutathione, and uric acid. SODs convert two superoxide anions
into one molecule of hydrogen peroxide and oxygen, and catalase catalyzes
the decomposition of hydrogen peroxide into water and oxygen, leading to
the removal of superoxide anion. Glutathione peroxidase plays an important
role as defense mechanism in mammals, birds and fish against oxidative
damage by catalyzing the reduction of a variety of hydroperoxides, using
glutathione as the reducing substrate. For example, glutathione peroxidase
reduces hydrogen peroxide to water by oxidizing glutathione (GSH) to its
oxidized form (GSSG), and the reduction of the oxidized form of glutathione
(GSSG) is then catalyzed by glutathione reductase. Through the glutathione
redox cycle, ROS is removed from cells. In addition to its role as a substrate
in GSH redox cycle, glutathione, as well as uric acid, also act as a direct
endogenous scavenger of hydroxyl radicals. Ascorbic acid scavenges free
radicals and reduces them into hydrogen peroxide. Hydrogen peroxide can
be further catalyzed by catalase to form water and oxygen. Alphatocopherol
can transfer a hydrogen atom with a single electron to free radicals, thus
removing the radicals before they can interact with cell membrane proteins
or generate lipid peroxidation(52).
1.2.5 Oxidative stress in atherosclerosis
Oxidative stress has been identified throughout the process of
atherogenesis, beginning at the early stage when endothelial dysfunction is
barely apparent(53). As the process of atherogenesis proceeds, inflammatory
cells, as well as other constituents of the atherosclerotic plaque release large
amounts of ROS, which further facilitate atherogenesis. In general, increased
production of ROS may affect four fundamental mechanisms that contribute
to atherogenesis: oxidation of LDL, endothelial cell dysfunction, vascular
SMCs, growth and monocytes migration(54).
A number of studies suggest that ROS oxidize lipids and that the
oxidatively modified LDL is a more potent proatherosclerotic mediator than
the native unmodified LDL(55). The suggestion is based on the observations
that high plasma levels of ox-LDL are present in patients with
atherosclerosis and that antibody to ox-LDL is detected in plasma of most
patients with atherosclerosis(56). Strong evidence in favor of a proatherosclerotic role for ox-LDL comes from a number of studies
demonstrating the noxious effects of ox-LDL on various components of the
arterial wall. For example, ox-LDL causes activation of the endothelial cells
lining the arterial wall, resulting in the expression of several adhesion
molecules that facilitate the adhesion of monocytes/macrophages (57). oxLDL also activates inflammatory cells and facilitates the release of a number
of growth factors from monocytes/macrophages(58,59). Vascular SMCs
exhibit intense proliferation when exposed to ox-LDL. ox-LDL enhances the
formation of matrix metalloproteinases (MMPs) in vascular endothelial cells
and fibroblasts(60) thus setting the stage wherein oxidative stress leads to
rupture of a soft plaque. In addition, ox-LDL upregulates the expression of
its endothelial receptor LOX-1 and other scavenger receptors mainly
expressed on macrophages/monocytes. The increased expression of these
receptors is responsible for the uptake of ox-LDL and the formation of foam
cells, which is an early step in atherogenesis(45).
1.3 Endothelium dysfunction
1.3.1 Endothelial dysfunction as an initial step of atherosclerosis
The endothelium is a flat monolayer of cells that cover vascular
lumina throughout the body. In adult, the number of endothelial cells is
approximately one trillion. The total surface area of the endothelium is as
large as six tennis courts and the weight of whole cells is larger than that of
the liver. In other words, the endothelium is the largest organ in the body(61).
Endothelial cells play diverse biological roles, such as maintaining
vascular tone and structure, regulating intravascular hemostasis and
permeability, and cell adhesion and migration(62). Recent progress in
vascular biology has revealed that the endothelium releases a large number
of vasoactive substances. These substances are divided into two classes:
endothelium-derived relaxing factors (EDRFs), and endothelium-derived
contracting factors (EDCFs). It has been shown that EDRFs such as nitric
oxide (NO). Endothelial Dysfunction as an Initial Step for Atherosclerosis
which caused by imbalance between EDRFs and EDCFs precedes and
promotes atherosclerosis by several mechanisms, such as adhesion of
monocytes and platelets;and increase in vascular permeability, and
proliferation and migration of smooth muscle cells. These processes result in
plaque formation and intimal thickening, whereas EDCF such as endothelin
have opposite effect and participate in the progression of cardiovascular
diseases. The exposure to coronary risk factors decreases the bioactivity of
EDRFs and increases the release of EDCFs(63,64). Thus, coronary risk factors
impair the bioactivity of NO derived from endothelial cells and the impaired
bioactivity of NO exacerbates atherosclerosis(65,66). Furthermore, it has been
shown that atherosclerosis by itself decreases biological NO activity, leading
to a vicious cycle(65).
1.3.2 Oxidative stress and endothelial dysfunction
Oxidative stress induced by reactive oxygen species impair the
biological activities of endothelium-derived NO through several
mechanisms. Endothelium-dependent vasodilation was lost at basal level and
was restored by anti-oxidant ascorbic acid in patients with mitochondrial
diseases, suggesting that oxidative stress may be involved in premature
cardiovascular diseases and anti-oxidant may become a therapeutic tool in
mitochondrial diseases. However, it remains unknown whether the
circulating form of oxidized LDL impairs endothelial function (67).
1.3.3
Antioxidant
properties
of
nitric
oxide
in
atherosclerosis
Nitric oxide has many physiological actions that can be interpreted to
be potentially antiatherosclerotic(68,69). It inhibits 1) platelet aggregation and
adherence to endothelial cells, 2) monocyte adherence to endothelial cells, 3)
the expression of the monocyte chemoattractant protein, 4) vascular smooth
muscle cell migration and proliferation and 5) the in vivo intimal
proliferative response to ballon injury. Nitric oxide reduces oxidant stress in
the vascular wall, which in turn may lower the rate of LDL oxidation and the
expression of redox-sensitive genes that contribute to atherogenesis. In fact,
vascular NO either suppresses the expression of adhesion molecules by
endothelial cells or the generation of products that are chemotactic for
monocytes such as oxidized LDL. There is accumulating evidence that the
salutary effects of NO are diminished in atherosclerotic vessels due to its
reactions with reactive oxygen species. In particular, the reaction of NO
with O2•-, as well as its reaction with LO. and LOO. to inhibit lipid
oxidation, suggests that NO can both enhance and inhibit lipoprotein
oxidation in the vessel wall. The removal of NO from the vascular
compartment by its rapid reactions with these free radical species will
concomitantly lower its steady state concentration, and hence increasing
platelet and inflammatory cell adhesion to the vessel wall and impairing
endothelial-dependent mechanisms of relaxation(70).
Nitric oxide has been observed to play a critical role in regulating
lipid oxidation induced by reactive oxygen and nitrogen species (71,72).
Nitric oxide and mechanisms underlying impaired vasomotor
responses in atherosclerosis. Endothelium plays an important role in
maintaining vascular integrity by the synthesis and release of vasoactive
substances such as NO. The changes that occur during atherosclerosis
include the loss of the control of vascular tone, a NO-dependent event. The
mechanisms accounting for endothelial dysfunction in hypercholesterolemia
have not been completely elucidated, but may be explained by decreased
bioavailability of NO due to either decreased expression of the eNOS,
decreased substrate availability, presence of an endogenous eNOS inhibitor
or increased NO degradation by reactive oxygen-and nitrogen species(73).
1.4 Treatment of Atherosclerosis
The treatment of atherosclerosis relies heavily on the reduction
of risk factors particularly elevated serum lipids level, hypertension, diabetes
and cigarette smoking. The incidence of cardiac and cerebral infarction can
be diminished considerably by controlling risk factors. Lifestyle factors are
also of greatest importance in the treatment of atherosclerosis(74,75).
1.4.1 Lifestyle change
The most common treatments focus on lifestyle changes to reduce
cholesterol and other problems that contribute to atherosclerosis. Dietary
modifications usually incorporate eating foods that are low in saturated fats,
cholesterol, sugar, and animal proteins. Foods high in fiber, such as fresh
fruits and vegetables, and whole grains, are encouraged. By consuming fruits
and vegetables, the person also consumes helpful dietary antioxidants, such
as carotenoids found in vegetable pigments, and bioflavenoids in fruit
pigments. Liberal use of onions and garlic is recommended, as well as eating
fish, especially cold-water fish, such as salmon. Smoking, alcohol, and
coffee are to be avoided, and exercise is strongly recommended(76).
1.4.2 Medications
Medications commonly prescribed in the treatment of atherosclerosis
include anti-hypertensives and cholesterol reducing drugs(77).
1.Anti-hypertensives
While anti-hypertensives do not control the process of atherosclerosis,
they are successful in controlling one of the primary side effects. These
drugs include calcium channel blockers, ACE inhibitors, and angiotensin
receptor blockers(78).
2. Cholesterol reducing drugs
Keeping serum cholesterol level in the normal range not only helps
prevent heart attacks and strokes but may also prevent the progression of
atherosclerosis(79). These drugs include statins, bile-acid–binding resins,
nicotinic acid, fibrates and cholesterol absorption inhibitors (ezetimibe) .
1.4.3 Therapeutic approach
It is hoped that understanding the biology of the endothelium in
atherosclerosis will lead to therapies that will retard its progression, and
reduce the incidence or consequences of acute complications. At present,
insights from endothelial biology have provided an explanation of the
mechanism of action of existing therapies (NO donors, aspirin), but new
treatments have been slow to emerge. Endothelin antagonists have been
developed for the treatment of pulmonary hypertension. However their
usefulness in the treatment of human atherosclerosis has been hampered by
toxicity. L-arginine supplementation can retard the progression of
atherosclerosis in animal models . Short-term studies suggest that Larginine
supplementation can restore conduit and resistance vessel endotheliumdependent dilatation in humans with risk factors for atherosclerosis. It is
possible that modulation of the BH4 pathway in humans may be a future
therapeutic target. Whether measures to preserve endothelial function may
reduce the incidence of acute vascular events, or limit tissue damage during
ischaemia-reperfusion injury, remains to be seen . Sildenafil, an inhibitor of
cGMP breakdown by phosphodiesterase V, has been a therapeutic advance
in the treatment erectile dysfunction caused by atherosclerotic disease, but
its potential for the treatment of the systemic vasculature is uncertain.
Therefore our study is undertaken to assess the effect of phosphodiesterae
inhibitor on systemic vasculature in experimental model of
atherosclerosis(approach)(80).
1.5 Phosphodiesterase Inhibitors
Phosphodiesterases (PDEs) are a superfamily of enzymes involved in
regulating intracellular signaling by cleaving cyclic adenosine
monophosphate (cAMP) or cyclic guanine monophosphate (cGMP) to their
corresponding’ nucleotides, and thus rendering them inactive. Cyclic AMP
is a ubiquitous second messenger that controls cell functions such as growth,
differentiation, survival and inflammation by affecting protein kinase
activators (PKA) in response to Gs or Gi coupled receptor activation. PDEs
act as a critical feedback mechanism in the cAMP cellular signaling cascade.
There are at least 11 PDE families recognized in mammals that have been
characterized with respect to biochemical properties, expression, and their
affects on other proteins or pathways. PDEs have become novel therapeutic
drug targets. They share common catalytic sequences but they differ in
substrate specificity, tissue distribution and expression, sensitivity to
inhibitors and mode of regulation(81).
1.5.1
Sildenafil
Sildenafil belongs to a class of compounds called selective PDE-5inhibitors(82). Sildenafil was synthesized by a group of pharmaceutical
chemists working at Pfizer's . It was initially studied for use in hypertension
and angina pectoris. The first clinical trials, suggested that the drug had little
effect on angina, but that it could induce marked penile erections(83). Pfizer
therefore decided to market it for erectile dysfunction by the US Food and
Drug Administration on March 27, 1998, becoming the first oral treatment
approved to treat erectile dysfunction in the United States, and offered for
sale in the United States later that year(84).
1.5.1.1 Mechanism of action and pharmacological properties
Sildenafil is a potent inhibitor of PDE-5 which is the predominant
isozyme that metabolizes cGMP in the corpora cavernosa of the penis.
cGMP is the second messenger of NO and a principal mediator of smooth
muscle relaxation and vasodilatation in the penis (82).
Sildenafil is a potent inhibitor of PDE-5, with favorable selectivity
(>1000-fold) for human PDE-5 over human PDE2 (in the adrenal cortex),
PDE3 (in smooth muscles, platelets, and cardiac tissue),and PDE4 (in the
brain and lung
lymphocytes) and moderate selectivity (>80 fold ) over
PDE1 (in the brain, kidney, and smooth muscle). Sildenafil is only 10-fold as
potent for PDE5 as for PDE6(82).
In addition, sildenafil possess direct muscle relaxant potential possibly
via inhibiting Ca(2+) influx through both receptor-operated and voltage
dependent Ca(2+) channels(85) and induce vasorelaxation that is associated
with increases in the phosphorylation of heat shock related protein 20
(HSP20) (86).
1.5.1.2 Effect on hemodynamics
The data from hemodynamic studies would suggest that sildenafil is a
modest vasodilator with effects on both the venous and arterial tree (87). It
possesses vasodilatory properties, which result in mild, insignificant
decreases in BP when taken alone (82). Sildenafil improves microcirculation
in patients with Raynaud’s phenomenon(88,89).. During exercise and recovery,
sildenafil does not cause clinically significant alterations in hemodynamic
parameters in men with CAD(90). Sildenafil produces a transient modest
reduction in systolic and diastolic blood pressures (BP). No significant
effects are observed on heart rate (HR) (82), while systemic BP did not change
after sildenafil administration (2 mg/kg), and the blood flow in a normal
coronary artery increased(91). Sildenafil if used regularly may be responsible
for small yet significant decrease in BP in diabetic hypertensive patients
with erectile dysfunction along with improvement in their sexual
dysfunction (92) . Sildenafil may be useful in treating patients with HF, PH,
and CAD(93). Also sildenafil has no direct effect on cardiac repolarization
(QT interval)(94,95). The combination of sildenafil with any agent acting as a
NO donor is contraindicated due to life-threatening hypotension and the
increment in ventricular tachycardia/ventricular fibrillation (VT/VF)
vulnerability in the normal right ventricles (RV)(96). It also causes a marked
increase in sympathetic activation, evident both at rest and during stressful
stimuli (97). It was found that sildenafil blunts systolic responses to ßadrenergic stimulation and has potent effects on hearts stimulated by ßadrenergic or pressure overloads (98). Sildenafil does not worsen exercise
capacity and exercise-induced myocardial ischemia (99), while it reduces
cardiovascular remodeling associated with hypertensive cardiomyopathy
(100)
. Sildenafil has a cardioprotective effect and anti-ischemic effect (101).
Sildenafil inhibits platelet activation in patients with CAD (102).
1.5.1.3 Pharmacokinetics and metabolism
Sildenafil is rapidly absorbed orally, with absolute
bioavailability of 40%. Sildenafil is highly selective for the cGMPhydrolyzing isoform 5 with a half-maximal inhibition (IC50) of PDE-5
activity at a concentration of 3.5 nmol/L, followed by IC50 values of 34 to
38 nmol/L for PDE 6 (in the retina) and 280 nmol/L for PDE 1. The cAMPhydrolyzing PDE 3 and 4 and the cAMP- and cGMP-hydrolyzing isoform 2,
as well as PDE 7 to 11,are inhibited by sildenafil with an IC50 of >2600
nmol/L(82).
Plasma concentrations peak within 30 to 120 minutes of oral
dosing in the fasted state. Sildenafil is primarily metabolized by the
cytochrome(cyt.)P450 3A4 hepatic microsomal isoenzymes,which convert it
to an active N-desmethyl metabolite. Sildenafil and its active metabolite are
both highly bound to plasma proteins (96%), and their terminal half-lives are
4 hours each. sildenafil is excreted as metabolites predominantly in the
feces. Plasma levels of sildenafil are increased in patients aged >65 years
and in patients with hepatic impairment, severe renal impairment, and
concomitant use of erythromycin , cimetidine, ketoconazole, fluvoxamine
and indinavir and grapefruit juice increases sildenafil bioavailability(82).
1.5.1.4 Adverse effects
Sildenafil has these adverse effects: headache, flushing, rhinitis , nasal
obstruction ,dizziness, hypotension, postural hypotension, dyspepsia ,burning
sensation, increased perception of light, and blurred vision , myalgias,
changes in serum creatine kinase, priapism,a transient prolongation of the
BT, intracerebral hemorrhage, gastric variceal bleeding, epistaxis, and
hemorrhoidal bleeding, tonic-clonic seizures, transient ischemic attacks,
strokes, and transient global amnesia,.hepatotoxicity, atrial fibrillation,
migraine, stroke, gallbladder disorders, lichenoid drug eruption ,toxic
epidermal necrolysis, MI, vestibular neuritis-like symptoms(82).
1.5.1.5 Drug interaction
Vasodilator actions of nitrates are amplified with concomitant use of
sildenafil within the first 24 hours(82,103).
Sildenafil is an inhibitor of the cyt. P450 2C9 metabolic pathway.
Therefore, administration of sildenafil could result in a significant increase
in the plasma concentrations of other drugs metabolized through this
pathway. sildenafil is predominantly metabolized by both the P450 2C9
pathway and the P450 3A4 pathway. Thus , cimetidine ,ciprofloxacin ,
clarithromycin ,erythromycin, itraconazole, nefazodone, diltiazem, ritonavir
and grapefruit juice increases the plasma concentrations of sildenafil
.Sildenafil should be used with caution in patients who take alphablockers,and nicorandil(103).
1.5.2 Vardenafil
Vardenafil is the second oral selective PDE-5 inhibitor for erectile
dysfunctin with the highest potency and minimal inhibition of other
PDEs(104). It was licensed in Europe in 2003 and in the USA in late 2003 (105).
Vardenafil is a PDE5 inhibitor used for treating impotence (erectile
dysfunction) that is sold under the trade name Levitra (Bayer AG, GSK, and
SP)( 106). Vardenafil's indications and contra-indications are the same as with
other PDE5 inhibitors. It is closely related in function to sildenafil (Viagra)
and tadalafil (Cialis). Structurally, the difference between the vardenafil
molecule and sildenafil is a nitrogen atom's position and the change of
sildenafil's piperazine ring methyl group to an ethyl group(106).
1.5.2.1
Mechanism
of
action
and
pharmacological
properties
The mechanism of action and pharmacological properties of sildenafil and
vardenafil are comprehensively alike.
1.5.2.2 Hemodynamic effects
Recent studies suggest that there are pharmacological differences
between the three available PDE5- inhibitors. Gofrani et al. compared the
short-term haemodynamic effects of sildenafil, vardenafil and tadalafil in a
well-defined population suffering from chronic pulmonary arterial
hypertension. Results showed that vardenafil lacked selectivity for the
pulmonary circulation, while only sildenafil improved arterial oxygenation.
In another study, Teixeira et al. demonstrated that vardenafil, but not
sildenafil or tadalafil, affects Ca2+ handling in the rat aorta besides
increasing cGMP levels(107). The ability to exercise among patients with
CAD is not impaired with vardenafil treatment (108).Vardenafil demonstrats
clinically significant differences (fainting) with respect to sildenafil only
during the first doses(109). Vardenafil produces a slight hypotensive effect
and a minor compensatory increase in heart rate(110). While, unlike sildenafil,
vardenafil has been associated with a slight prolongation of the QT interval
(111)
. However, the co-administration of vardenafil with tamsulosin is not
associated with clinical significant hypotension (112). The study by Deibert
et al., showed that vardenafil (another phosphodiesterase type-5 inhibitor)
was found to lower portal pressure in four of five patients with Child A
cirrhosis(113). In investigating the effects of vardenafil, on myocardial and
endothelial functions, it was conclude that the myocardial contractility
parameters showed no significant changes when compared to that at baseline
in addition to an initial drop of blood pressure up to 15—20%(114). Vardenafil
induces a protective effect against ischemia/reperfusion injury(115), and
specifically relaxes coronary resistance vessels(116). However, Thadani et al.
,studied men with reproducible angina on exercise and showed that
vardenafil 10 mg did not change the total exercise time or time to angina
compared with placebo and actually increased the time to ischemic STsegment changes(117). While Vardenafil has no effect on the
pharmacodynamics of warfarin (prothrombin-time),and aspirin (bleeding
time)(118).
1.5.2.3 Pharmacokinetics
Vardenafil needs lower absolute concentrations to inhibit PDE-5.
However, relative selectivity for PDE-5 is similar to that of sildenafil(119). It
shares the molecular structure of sildenafil(120). The time to peak serum
concentration of vardenafil is 0.75 hour comparable to that of sildenafil
(1.16 hours).It is metabolized by the liver and excreted mainly in the feces
with an excretion half-life of 4.7 hours, only longer than that of
sildenafil(approximately 4 hours) and rapid onset of action. It can be taken
after eating a moderately fatty meal.Its potency was approximately 25 times
greater than that of sildenafil and 48 times greater than that of tadalafil while
a high-fat meal may alter Cmax slightly and delay the absorption up to 1
hour, a moderate-fat meal has no effect on vardenafil pharmacokinetics.
Ritonavir can affect its hepatic metabolism(121).
1.5.2.4 Side effects
Vardenafil has these adverse effects: headaches, flushing, rhinitis,
dyspepsia, epileptic seizures, atrial fibrillation, with less visual than
sildenafil changes, and urticaria(122,123).
1.5.2.5 Drug interaction
Vardenafil may potentiate the hypotensive effects of nitrates(123); its
lack of interaction at 24 hours(120). Vardenafil increases the hypotensive
effect of α-adrenergic blocking drugs except tamsulosin(124).
Briganti et al. (2005 ), (121) showed that ritonavir, indinavir,
erythromycin, itraconazole, and ketoconazole reduce vardenafil clearance.
1.6 Doppler Ultrasound Technique
Duplex and triplex U.S. techniques are non-invasive diagnostic
techniques that obtain flow velocity data and used as an alternative to
invasive intra-arterial angiography which is associated with local and
systemic complications, and to acquire direct physiologic information about
affected arteries. Duplex U.S. combines a B-mode ultrasound image with a
pulse wave Doppler unit; whereas triplex U.S combines a B-mode
ultrasound image with a color flow unit and a pulse wave Doppler unit(125).
The advantages of duplex and triplex U.S. include absence of
complications, relatively low costs, and widespread availability(126). These
techniques provide an accurate evaluation of hemodynamically significant
occlusive vascular lesions. They are used in the evaluation of the extracranial carotid arteries, iliofernoral arteries, abdominal arteries and
hemodialysis shunts. These systems obtain information regarding blood
flow velocity, lumen area, plaque composition and ulceration, and
hemodynamic turbulence(127).
With duplex and triplex U.S. techniques, the severity of a stenosis
can be determined by using peak systolic velocity (PSV) measurements in
arteries with reduced luminal diameter, the PSV ratio at the site of the
stenosis and the adjacent normal artery, the end-diastolic velocity and other
less firmly established criteria(128).
1.6.1 Doppler parameters and doppler indices
 Peak systolic velocity (PSV)
Peak systolic velocity is a measure of the maximum velocity of blood
flow during systole. Using Doppler principles, as an artery narrows, the
velocity of blood flow increases(129). So the peak systolic velocity is used as
a criteria for diagnosis of arterial stenosis, e.g. the PSV in the renal artery is
less than 180 cm/s in normal adult human, an increase in peak systolic flow
velocity greater than 180 cm/s indicates a renal artery stenosis of more than
50% diameter reduction(130).
 End diastolic velocity (EDV)
End diastolic velocity is a measure of the velocity of blood flow at the
end of diastole. End diastolic velocity is a measure of resistance in the
distal arteries, where high resistance in the distal vessels produce low
diastolic flow in the supplying artery(131).
 Mean velocity
Mean velocity is the average blood flow velocity during the cardiac
cycle. Sometimes mean velocity is used together with the vessel crosssectional area to calculate blood flow rate. However, it is difficult to
measure mean velocity accurately and there are several other problems
associated with calculating flow rate(132).
Resistive index (RI) and Pulsatality index (PI)
Resistive index (RI) is a pulsed-wave Doppler index used for
measurements of downstream resistance in arteries (high resistance in the
distal vessels produce low diastolic flow in the supplying arteries and result
in a high value for this index and vise versa)(131).
Pulsatality index ( PI) is also a pulsed-wave Doppler index designed
to quantify the pulsatality or oscillations of the blood flow waveforms(131).
Originally both PI and RI were introduced to detect peripheral
vascular diseases, but they are rarely used in these conditions(133). However,
if the indices are measured in renal arteries, they are reported as reliable
measurements of down-stream renal resistance(134). An increasing amount of
information is available about the usefulness of PI and RI in the
investigation and monitoring of renal artery stenosis(135,136).
Ohta et al. (2005) reported that increased RI and PI of the renal
arteries is associated with arterial stiffness and reflects the severity of
systemic atherosclerosis(137). RI and PI are calculated via soft wave programs
built in all recent Doppler machines.
 Renal artery-to-aortic PSV ratio (RAR)
Renal-aortic ratio is the ratio of the PSV in the renal artery to the PSV
in the aorta . This Doppler index is used as a criteria for diagnosis of renal
artery stenosis. In adult human the normal value of renal-aortic ratio is less
than 3.5, RAR value equal to or more than 3.5 indicates a 60% renal artery
stenosis or more(129).
1.7 Blood Flow Rate and Blood Flow Velocity
1.7.1 Definition of flow rate and flow velocity
When considering flow in a system of tubes, it is important to
distinguish between velocity which is displacement per unit time (e.g. cm/s)
and flow rate which is volume per unit time ( e.g. ml/s)(138).
1.7.2 Factors affecting on blood flow and blood flow velocity
There is a linear relationship between blood flow rate and peak blood
flow velocity in fixed diameter vessels with laminar flow. So with the
exception of the effect of radius ( diameter ) of blood vessel on blood flow
rate, any other factor which increase or decrease blood flow rate will
increases or decreases the blood flow velocity(139).
The factors influencing on blood flow rate are explained by poiseuilles law :
F = P  r 4 / 8  l.
In which F is the rate of blood flow, P is the pressure difference between
the ends of the vessel, r is the radius of the vessel, l is the length of the
vessel and  is the viscosity of the blood(140).
So factors affecting on blood flow are:
1.Vessel radius
The blood flow rate is directly proportional to the fourth power of the
radius of the vessel which demonstrates the diameter of blood vessel (which
is equal to twice the radius). So as the vessel radius is increased the blood
flow rate increased. But there is inverse relationship between vessel radius
and blood flow velocity(138).
2.Viscosity :
As the blood viscosity increased the blood flow rate and blood flow
velocity are decreased, so the factors affecting on blood viscosity will affect
on blood flow rate and velocity. The most important factor is the
hematocrite. Gruber et al. (1999), and Rosenkrantz et al. (1984) found
that blood flow rate and velocity are increased in case of anemia and
decreased in case of polycythemia and dehydration (141,142). Blood viscosity
increases when blood temperature decreases. However, the increase in
viscosity is observed mostly at temperature below 15°C. So when blood
temperature is reduced, the blood flow rate and velocity are reduced(143).
3. Pressure gradient P
As the pressure gradient increased, the blood flow rate is increased.
Factors affecting on pressure gradient are cardiac output, peripheral
resistance, and vessel compliance(144).
1.7.3 Effect of atherosclerosis on blood flow velocity
Healthy blood vessels are elastic and flexible (compliant) (145).
Atherosclerosis is a form of the disease that causes blood vessels to become
thick and stiff because of fatty plaques that develop inside of arterial
walls(146). When atherosclerosis first begins, the fatty plaque grows and
begins to protrude into the blood vessel wall and restricts the amount of
blood that can flow past it. This type of blockage is called a stenosis. The
newly blocked vessel now has the same amount of blood trying to flow
through a smaller space. This causes an increase in the velocity of the blood
flow at the point where the stenosis is located(147,148).
1.7.4 Aortic blood flow
The normal Doppler wave forms of the aorta varies with the location.
In the upper aorta, there is a narrow well defined systolic complex with
forward flow during diastole. Below the renal arteries the diastolic flow is
much reduced and above the bifurcation it is absent or reversed(149). The
main abnormalities affecting the aorta are atheroma and aneurysm.
Atheroma can affect the aorta and produce stenosis or occlusion. In such
cases velocity ratios taken from above and at the stenosis can be used to
assess the degree of hemodynamic compromise, if there is significant
increase in the blood flow velocity and velocity ratio by a factor of two or
more, it would indicate a sever aortic stenosis(150).
1.7.5 Renal artery blood flow
To a large extent, the excretory and regulatory processes of the kidney
depend on the blood supply to the kidney. Therefore, it is not surprising that
it receives the highest blood flow per gram of organ weight in the body. The
combined blood flow through both kidneys is about 1100 ml/min which is
about 22% of the total cardiac output. Thus, a very substantial portion of the
total cardiac output flows through the kidneys(151).
The normal Doppler wave forms of the renal arteries in normal adult
human are characterized by a rapid systolic upstroke which is occasionally
followed by a secondary slower rise to peak systole and there is subsequently
a gradual diastolic decay but with persistent forward flow in diastole.
In normal adult human, the peak systolic velocity of the normal renal
artery is less than 180 cm/s(152).
1.7.6 Intra-renal arteries blood flow
The renal arteries branches into segmental arteries which subdivide
into interlobar arteries. Each interlobar artery penetrates the kidney through
a column of Bertin, before reaching the cortical surface divides into arcshaped arcuate arteries.
The normal Doppler wave forms of segmental arteries in normal adult
human at rest are pulsatile, diphasic, and characterized by sharp rapid
upstroke, having double systolic peak, and of high diastolic flow(153).
In normal adult human at supine position, the interlobar intra-renal
arteries peak systolic velocity and end diastolic velocity are in the range of
32-58 cm/s and 13-18 cm/s respectively(154).
1.8 Aim of the Study
This study was undertaken to evaluate the effects of sildenafil and
vardenafil on atherosclerosis progression.
2.1 Materials
The chemicals, drug and instruments used in the present study with their
supplier listed in the following tables
Table (1): list of chemicals pharmaceuticals, reagent and their supplier
Pharmaceuticals and reagents
Supplier
Sildenafil
pfizer,USA, Batch No 89R001E
Vardenafil
Bayer AG,Germany. Batch
no.NBRYO6.
pure cholesterol powder ( C27H46O)
M. wt = 386.67
BDH Chemicals Ltd Poole England.
Total cholesterol ,Triglyceride and
HDL Reagents
BIOLABO SA ,02160MAIZYFRANCE
Reduced Glutathione (GSH)
Biochemical
Trichloroacetic acid (TCA)
Merk, Germany
5,5-dithiobis(2-nitrobenzoic acid)
DTNB
Sigma
Ethylene Diamineteteraacetic acid
Disodium(EDTA)
BDH,U.K
Trishydroxymethylene
Merk, Germany
Absolute Methanol
Fluka
Thiobarbaturic acid (TBA)
Merk Co.Ltd
Table (2): Instrument and their supplier
Equipment
Company
Sensitive electrical balance
Sartorius /Germany
pH meter
Inolab /Germany
spectrophotometer
Shimadzu UV-1650(UV-visible)/japan
spectrophotometer
Apple UK
Vortex (super mixer)
LAB-Line /USA
Centrifuge
Hettich/Germany
Water bath
Memmert /Germany
2.2 Animals and study design
24 local domestic rabbits were used in this study. Their weight ranged
between 1.4-1.7kg, they were housed in the animal house of Al Kufa
College of medicine. They were kept in cages under a 12-h light:12-h dark
cycle at room temperature 25°C, and humidity was kept at 60–65%. , and
they were allowed to drink tap water and given standard chow diet at
libitum. During 1 wk of adaptation; alfalfa and other grasses or vegetables
were given but with small amounts until the end of this week when they
were prevented completely to avoid their influence on hypercholesterolemic
atherosclerosis protocol. After the 1st week of acclimatization the rabbits
were randomized into four groups as follow:
i.
Normal control group: This group consists of 6 rabbits; all rabbits
of this group were kept on standard chow diet and tap water
throughout the duration of study (12 weeks).
ii.
High cholesterol diet control group: (induced untreated group):
This group consists of 6 rabbits; all rabbits of this group were kept on
atherogenic diet (a 2% cholesterol-enriched diet) and tap water
throughout the duration of study (12 weeks).
iii.
Sildenafil treated group: This group consists of 6 rabbits. All rabbits
in this group were kept on atherogenic diet ( 2% cholesterol enriched
diet) and tap water for 6weeks, then they were treated with sildenafil 5
mg./kg. body weight / day orally(155) in a single dose at evening for the
next 6 weeks . The treatment continued together with the atherogenic
diet.
iv.
Vardenafil treated group: This group consists of 6 rabbits. All
rabbits in this group were kept on atherogenic diet ( 2% cholesterol
enriched diet) and tap water for 6weeks. Then they were treated with
vardenafil 18 mg./kg. body weight / day orally(156) in a single dose at
evening for the next 6 weeks. Also the treatment continued together
with the atherogenic diet.
 The standard diet for rabbits consisted of 10% wheat, 40% grass powder,
12% soybean cake, 20% corn, 10% wheat bran, 3% fish flour, 1% salt,
3% bone meal, and 1% multivitamins (percent by weight)(154).

The high-cholesterol experimental diet consisted of 2% cholesterol was
made by 20gm of powder cholesterol for each 1000gm of standard rabbit
diet.
 The food intake was recorded every day. The rabbit weigh weekly to
ensure proper food consumption among all groups.
 The experimental induction of atherosclerosis were conducted in
accordance with Meena Narayanasawamy, Kenneth C. Wright,
Krishna Kandarpa (2000)(158) that use 2% cholesterol for 12 week as
model of hypercholesterolemic atherosclerosis in rabbit.
2.3 Drugs
2.3.1 sildenafil
Sildenafil used in dose of 5 mg / kg. A tablet dissolved in 15 cc DW
,in concentration of 5 mg/ 1.5 cc and the dose given according to body
weight orally in a single dose at morning .(Sildenafil solubility in D.W. is
3.5 mg/1 cc)(156).
2.3.2 vardenafil
Vardenafil used in dose of 18 mg/kg has been used .A tablet
dispersed in tap water and the dose given according to the body weight by
oral gavages(159).
2.4 Preparation of sample
2.4.1 Blood sampling
From each rabbit about 3 ml of blood was collected from the central ear
artery without user heparin after an overnight fasting. The blood sampling
was done firstly at the start of the study, i.e. at zero time, 6 weeks of the
induction period, i.e. at the start of treatment with sildenafil and vardenafil,
and then every week of the treatment course. The blood samples were
allowed to clot at 37 C and centrifuged at 3000 rpm for 15 min. , Sera were
removed, and analyzed for determination of serum total cholesterol,
triglycerides, HDL- C and oxidative stress parameter (MDA,GSH).
2.4.2 Tissue sampling
At the end of the protocol (12 weeks on their respective diets), rabbits were
sacrificed with high dose of Phenobarbital sodium (200mg, intravenously).
The killed rabbits were dissected through the chest wall to make the aorta
accessible for resection. The aortic arch was exteriorized, cleaned of
adherent fat and connective tissue excised. All specimens were
immediately fixed in 10% formaldehyde solution. After fixation they were
processed in usual manner. The sections were examined by microscope
under magnification power of (×10 and ×40) then the histological changes
were determined according to American Heart Association classification of
atherosclerosis phases(160,161)
which divides atherosclerotic lesion into
different degree and phases as follow:
 Initial Lesion
Type I: increase the numbers of macrophage and appearance of Foam
Cells distributed at random
Type II: (fatty streak) lesion: consist primarily of layers of macrophage
foam cells and lipid-laden smooth muscle cells and include lesions
grossly designated as fatty streaks
 Intermediary lesion (preatheroma)
Type III: describe as a pathological intimal thickening with a poorly
formed fibrous cap (because of the absence of a necrotic core). Typically,
these lesions show incompletely coalesced extracellular lipid, most of
which is located deep within the plaque, underneath a layer of
macrophages and smooth muscle cells.
 Advance lesion
Type IV: (atheroma) lesion: confluence of lipid collections creates an
extracellular dense accumulation of fat in a well determined area of
tunica intima in other word "a well-formed cellular cap overlying a
confluent, necrotic, fatty core".
Type V: (fibro-atheroma) lesion: thick layers of fibrous connective tissue
(thick cellular caps) overlying a largely necrotic, fatty mass.
 Complicated lesion
Type VI: complicated fibro-atheroma lesion: plaque with surface defect
and/or hematoma/hemorrhage and/or thrombotic deposit
2.5 Measurement of samples
2.5.1 Measurement of serum total cholesterol(162)
Reagents used are supplied by BIOLABO SA and the method is as
following : Mix Reagent 1 ( buffer ) + Reagent 2 ( enzymes ), mix gently
until complete dissolution ( approximately 2 min.).
Reagent
Blank
Standard
Assay
1 ml
1ml
1ml
–
–
Demineralised water 0.1 ml
Standard
–
0.1 ml
–
Specimen
–
–
0.1 ml
Wait for 10 min. and then record absorbencies at 500 nm against reagent
blank.
Calculate the result as follows :
Result =
𝐴𝑏𝑠 (𝐴𝑠𝑠𝑎𝑦)
𝐴𝑏𝑠 (𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑)
× 200 ( Standard concentration)
Composition of Reagents :
R1 (Buffer) composed of the following:
Phosphate buffer, Chloro-4-phenol, Sodium Cholate, Triton×100, and
preservatives
R2 ( Enzymes) composed of the following:
Cholesterol oxydase (CO), Cholesterol esterase (CE), Peroxydase (POD), 4Amino-antipyrine (PAP) and PEG 6000)
R3( standard) contains:
Cholesterol 200mg./dl
Principle(163):
The principle is described by Allian C., the reaction scheme is as
follow:
Cholesterol esters →
Cholesterol + O2 →
𝐶𝐸
𝐶𝑂
Cholesterol + free fatty acids
Cholesten-4-one-3 + H2O2
2H2O2 + phenol + PAP →
𝑃𝑂𝐷
Quinoeimine (pink) + 4 H2O
The absorbance of the colored complex (Quinoeimine), proportional to the
amount of cholesterol in the specimen, is measured at 500nm.
2.5.2 Measurement of Triglycerides Concentration(164)
Reagents used are supplied by BIOLABO SA and the method is as
following : Mix Reagent 1 ( buffer ) + Reagent 2 ( enzymes ), mix gently
until complete dissolution ( approximately 2 min.).
Reagent
Blank
Standard
Assay
1 ml
1ml
1ml
–
–
Demineralised water 0.1 ml
Standard
–
0.1 ml
–
Specimen
–
–
0.1 ml
Wait for 10 min. and then record absorbencies at 500 nm against reagent
blank.
Calculate the result as follows :
Result =
𝐴𝑏𝑠 (𝐴𝑠𝑠𝑎𝑦)
𝐴𝑏𝑠 (𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑)
× 200 ( Standard concentration)
Composition of Reagents:
R1 (Buffer) composed of the following :
Pipes, Mgnesium chloride, Chloro-4-phenol, and preservatives
R2 ( Enzymes) composed of the following:
Lipase, Peroxydase (POD), Glycerol-3-phosphate oxydase (GPO), Glycerol
Kinase (GK), 4-Amino-antipyrine (PAP), and Adenosine triphosphate Na
(ATP)
R3( standard) contains:
Glycerol equivalent to Triglycerides 200mg./dl
Principle(165):
The principle is described by Fossati and Prencipe. The Reaction
scheme is as follow
Triglycerides →
𝐿𝑖𝑝𝑎𝑠𝑒
Glycero+ ATP ⇔
𝐺𝐾
glycerol + free fatty acids
glycerol-3-phosphate + ADP
𝐺𝑃𝑂
Glycerol-3-phosphate + O2 ⇔
Dihydroxyacetone Phosphate + H2O2
𝑃𝑂𝐷
H2O2 + 4-Chlorophenol + PAP →
Quinoeimine (pink) + H2O
The absorbance of the colored complex (Quinoeimine), proportional to the
amount of triglycerides in the specimen, is measured at 500nm.
2.5.3 Measurement of Serum HDL-C Concentration(166)
The used reagents were supplied by Spinreact and the method as the
following:
Precipitation:
1-The following solutions are added into a centrifuge tube:
R (µ L)
100
Sample (ml)
1.0
2- Solutions are mixed well, allowed to stand for 10 minutes at room
temperature.
3- Centrifugation at 4000 rpm for 20 minute.
4- Then the supernatant is collected to test HDL-C.
Test:
HDL-C is measured by following the cholesterol reagent instructions.
Calculations :
With calibrator :
𝐴𝑏𝑠 (𝑆𝑎𝑚𝑝𝑙𝑒)
𝐴𝑏𝑠 (𝐶𝑎𝑙𝑖𝑏𝑟𝑎𝑡𝑜𝑟)
× (Calibrator conc.) = mg/dl HDL-C in the sample.
With factor :
(Abs) sample x 320 = mg/dl HDL-C in the sample.
Composition of Reagents :
R
Phosphotungstic acid
14 mmol/L
Precipitating Reagent
Magnesium chloride
2 mmol/L
Optional
Cholesterol
Ref. 1001092
Ref. 1001093
Principle(166):
The VLDL and LDL lipoproteins from serum or plasma are
precipitated by phosphotungstate in the presence of magnesium ions. After
removed by centrifugation the clear supernatant containing HDL is used for
the determination of HDL cholesterol
2.5.4 Calculation of LDL, VLDL and Atherogenic index(77)
LDL = Total cholesterol – (HDL + VLDL)
VLDL = serum triglyceride / 5
Atherogenic index = (Total cholesterol – HDL-C)/HDL-C.
2. 5.5 Measurement of serum malondialdehyde (MDA) Level(167).
The level of malonaldialdehyde was determined by modified procedure
described by (Guidet B.and Shah S.V.,1989)
Principle :
The test is based on reaction of MDA with thiobarbituric acid (TBA);
forming an MDA-TBA2product that absorb strongly at 532nm as follow
Preparation of reagent:
1. Trichloroacetic acid reagent 70%
70gm of TCA was dissolved in a final volume of 100ml of DW
2. TCA reagent 17.5%
5ml of 70% was diluted upto 20ml with distall water
3. TBA reagent 0.6%
0.06gm of TBA was dissolved in final volume of 10ml of DW using a water bath
for complete dissolving of TBA 1
Procedure :
1- 150ul of serum sample was poured in a test tube and 1ml of 17.5% TCA was
added
2- 1ml of 0.06% TBA was added
3- The tube were mixed well by vortex ,incubated in boiling water bath for 15min,
and then allow to cool
4- 1ml of 70% TCA was added
5- The mixture was left to stand at room temperature for 20min
6- The tube was centrifuge at 2000Xg for 15min ,and the supernatant was taken
out for measuring spectrophotometriclly at 532nm
Calculation of serum MDA:
The concentration of MDA =
𝑎𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒
×𝐷
𝐿∗𝜀
L: Light bath (cm)
𝜀: Extinction coefficient =1.56 × 105 M-1 Cm-1
D: Dilution factor =
𝑣𝑜𝑙𝑢𝑚𝑒 𝑢𝑠𝑒𝑑 𝑖𝑛 𝑅𝑒𝑓.(𝑚𝑙)
0.15
=
1+1+1+0.15
0.15
= 21
2. 5.6 Determination of serum reduced glutathione (GSH)
Principle(168):
5,5-dithiobis (2-nitrobenzoic acid) DTNB is a disulfide chromogen,
that is, readily reduced by sulfahydryl group of GSH to intensely yellow
compound . The absorbance of reduced chromogen is measured at 412nm
directly proportional to the GSH concentration
Preparation of reagents:
1- Precipitating solution (TCA 50%)
50gm of TCA were dissolved in final volume of 100ml of DW.
2- Ethylenediamine tetra-acetic acid-disodium (EDTA-Na2)(0.2M)
3.7224gm of EDTA-Na2 was dissolved in final volume of 50ml DW.
3- Tris –EDTA Buffer 0.4845gm of tris was dissolved in 8ml of DW. 1ml
of 0.2 M EDTA-Na2 solutions was added and brought to final volume of
10ml with DW. The PH was adjusted to 8.9 by the addition of 1M of
HCL .this solution is stable for at least 10days.
4- DTNB reagent (0.01M)
0.0396 g of DTNB was dissolved in absolute methanol, and brought to
the final volume of 10ml .this solution is stable for 13 week at 4C
5-GSH standards solution (0.001M)
Stock standards solution (0.001M) was prepared by dissolving
0.0156gm of GSH in final volume of 50ml of 0.2M EDTA solution,
dilutions were made in EDTA solution to 5.10,25,50,100,200,300
&400uM
Procedure:
Duplicate of each standard and sample test tubes were prepared.
Solution were mixed as in the following:
Reagent
Sample (uL)
Reagent blank(uL)
Standard (uL)
Serum
100
-
-
Standard
-
-
100
DW
800
900
800
TCA
100
100
100
Tube were mixed in vortex mixer intermittently for 10-15min,and
centrifuge for 15minat 3000xg,then pippetted into tubes.
Reagent
Sample (uL)
Reagent blank(uL)
Standard (uL)
Supernatant
400
400
400
Tris-EDTA
800
800
800
DTNB reagent
20
20
20
Tubes were mixed in a vortex mixer. The spectrophotometer was
adjusted with reagent blank to read zero absorbance (A) at 412nm and the
absorbance of the standards and sample was read within 3min of the addition
of the DYNB reagent
Calculation of serum GSH:
The concentration of serum GSH is obtained from the calibration curve in
uM (figure )
abs
0.25
y = 0.0005x + 0.0089
R² = 0.9852
Absorbance
0.2
0.15
0.1
0.05
0
0
100
200
300
400
500
GSH Concentration uM
2.6 Measurement of aortic diameter, intima-media thickness
and blood flow velocities of the aorta, renal artery and
intra-renal arteries
A triplex Doppler ultrasound machine (Siemens versa plus, made in
Germany) which combines a B-mode ultrasound image with a pulse wave
and color flow Doppler unit was used for examining rabbits. A 7.5 – 10
MHz linear array probe was used for this purpose. All rabbits were examine
in private clinic by Dr. Akeel AM. H. Zwain PhD U.K. Lecturer in
Cardiovascular Physiology. This machine was used for measuring aortic
diameter and intima-media thickness and aortic, renal artery and intra-renal
arteries blood flow velocities at the start of the study (at zero time). 4 rabbits
were examined after 6 weeks for follow up ( i.e. when starting the treatment
with sildenafil and vardenafil ), and again all rabbits were examined at the
end of the study (after 12 weeks).
The left lateral side of all rabbits was shaved by an electrical clipper.
The rabbits were examined at morning after an overnight fasting to prevent
the occurrence of intestinal gases which may prevent optimal visualizing of
the arteries; the left lateral side was used for technical reasons . Rabbits
were sedated by administration of 5mg/kg body weight diazepam intraperitoneally(169). Doppler angle was set at zero, the aorta was first visualized
and identified, the diameter and intima- media thickness of the aorta ( at
the origin of renal artery ) were measured. Then the aortic blood flow
velocities ( peak systolic velocity and end diastolic velocity ) were
measured electronically by the soft wave device built in the Doppler
machine. The renal artery and intra renal arteries of the left kidney were
visualized and identified using color flow mapping and their blood flow
velocities ( peak systolic velocity and end diastolic velocity ) were also
measured as mentioned earlier.
2.7 Calculation of Doppler indices(170)
The Doppler indices calculated include the following
1-Calculation of resistive index ( RI):
Resistive index (RI) was calculated from the following equation:
RI =
(𝑝𝑒𝑎𝑘 𝑠𝑦𝑠𝑡𝑜𝑙𝑖𝑐 𝑣𝑒𝑙𝑜𝑐𝑖𝑡𝑦(𝑃𝑆𝑉)−𝑒𝑛𝑑 𝑑𝑖𝑎𝑠𝑡𝑜𝑙𝑖𝑐 𝑣𝑒𝑙𝑜𝑐𝑖𝑡𝑦)
𝑝𝑒𝑎𝑘 𝑠𝑦𝑠𝑡𝑜𝑙𝑖𝑐 𝑣𝑒𝑙𝑜𝑐𝑖𝑡𝑦(𝑃𝑆𝑉)
2-Calculation of pulsatality index ( PI):
Pulsatality index (PI) was calculated from the following equation:
PI =
(𝑝𝑒𝑎𝑘 𝑠𝑦𝑠𝑡𝑜𝑙𝑖𝑐 𝑣𝑒𝑙𝑜𝑐𝑖𝑡𝑦(𝑃𝑆𝑉)−𝑒𝑛𝑑 𝑑𝑖𝑎𝑠𝑡𝑜𝑙𝑖𝑐 𝑣𝑒𝑙𝑜𝑐𝑖𝑡𝑦 )
𝑚𝑒𝑎𝑛 𝑣𝑒𝑙𝑜𝑐𝑖𝑡𝑦
The mean blood flow velocity (MV) is calculated from this equation(171) :
MV = 1/3[peak systolic velocity +2(diastolic velocity)]
3-Calculation of renal-aortic ratio (RAR):
This is the ratio of the peak systolic velocity (PSV) in the renal artery to the
peak systolic velocity (PSV) in the aorta. So it is calculated by the following
equation:
RAR =
𝑃𝑆𝑉 𝑖𝑛 𝑟𝑒𝑛𝑎𝑙 𝑎𝑟𝑡𝑒𝑟𝑦
𝑃𝑆𝑉 𝑖𝑛 𝑎𝑜𝑟𝑡𝑎
2.8: Histopathological procedure
The aortic arch with about 1 cm of the descending aorta was fixed in 10%
formalin for 24 hour, then washed and dehydrated in ascending series of
ethanol solution (70,80,90,95%) for 2hr. in each concentration and 4hr
divided on two changes for 100% (v/v) , cleared in two changes of xylen, 15
minutes for each, then embedded in paraffin wax. Thin section about (6mm)
was dewaxed in xylen for 6 minutes, hydrated in descending series of
ethanol (2 min. for each 2 change in 100% ethanol, then 2 min. in each of
95, 90, 80 and 70% v/v of ethanol solution, then transferred to distilled
water for 2 minutes. The sections were stained in haematoxylin for 2-5 min,
washed in tap water for 2-3 min, discolored in 0.5-1% HCL in 70% alcohol
for few seconds, washed in tap water for at least 5 min and stained in 1%
aqueous eosin for 1-2 min., then the section washed in tap water and
dehydrated in ascending series of ethanol ( 70,80,90,and 100% v/v ) for the
same period of hydration, then cleared in xylen and mounted in DPX (172).
2.9: Statistical analysis
Statistical analyses were performed using SPSS 12.0 for windows.lnc.
Data were expressed as mean ± SEM.; paired t-test was used to compare
the mean values within each group at different time. Analysis of Variance
(ANOVA) was used for the multiple comparison among all groups followed
by post-hoc tests using LSD method. Pearson correlation coefficient
was
used to assess the associations between two continuous variable of study
parameter. Chi-square test was also used to compare histopathological
changes in various groups. In all tests, P< 0.05 was considered to be
statistically significant(173).
3.1 Effect on serum total cholesterol (TC) level
In all groups of rabbits, the initial values of Serum TC were similar.
For the first 6 weeks, serum levels of TC were significantly increased in
groups (II, III, IV) fed with atherogenic diet(P<0.05) and remained
unchanged in the Normal control group (I), as shown in table (3) and figure
(1).
After another 6 weeks on which groups (II, III, IV) continued to have
cholesterol enriched diet, rabbits developed severe hypercholesterolemia
with serum levels of TC further increased significantly (P<0.05). The
changes in the levels of serum TC in the four groups are summarized in table
(3) and figure(1).
There was a statically significant difference(P<0.05) in serum levels
of TC between the normal control group (I) and the groups on high
cholesterol diet (II,III &IV). While The TC values showed insignificant
difference between the induced untreated group (II),vardenafil treated group
(III) & sildenafil treated group (IV), as shown in table (4).
Table (3): Changes of serum TC level in mg/dl of the four experimental
groups.(N = 6 rabbits in each group). The values are expressed as mean
±SEM.
At zero time
At 6 weeks
At 12 weeks
Normal control
48±2.1
50±2.6
49.4±2.3
Dietary induced untreated
47± 1.3
470±5.6*
710±9.4*
Vardenafil 18mg./kg
46±1.7
510±4.5*
690±5.8*
47.5±2.2
495±3.5*
745±6.4*
Sildenafil 5mg./kg
* p<0.05
Table (4) : Multiple comparisons between different groups mean values of
serum TC level in (mg/dl) using using ANOVA test
Groups
Vardenafil
Sildenafil
Induced untreated
–640.6*
–695.6*
Induced untreated
20.0
-35.0
Sildenafil
55.0
Normal control
* p<0.05
–660.6*
normal
dietary induced untreated
vardenafil
sildenafil
800
700
serum TC mg/dl
600
500
400
300
200
100
0
0 wk
6 wk
8 wk
10 wk
12 wk
Figure (1): Effect of Vardenafil and Sildenafil on serum TC level in comparison
to the two control groups (normal and induced untreated)
3.2 Effect on serum triglyceride (TG) level
The basal levels of serum TG were similar in all groups of rabbits. But it
increased significantly in groups (II, III, IV) fed with atherogenic
diet(P<0.05) for the first 6 weeks, while unchanged in the normal control
group (I), as shown in table (5) and figure (2).
After 12 weeks, there was an increase in the serum TG level (P<0.05) in
the induced untreated group, sildenafil treated group and vardenafil treated
group which continued to have cholesterol enriched diet, as shown in table
(5) and figure(2).
Serum levels of TG showed significant difference(P<0.05) between the
normal control group (I) and the groups on high cholesterol diet (II,III &IV).
The TG values were not significantly different between the untreated high
cholesterol diet (II) , vardenafil treated group(III) & sildenafil treated group
(IV), as shown in table (6).
Table (5): Changes of serum TG level in mg/dl of the four experimental
groups.(N = 6 rabbits in each group). The values are expressed as mean
±SEM.
At zero time
At 6 weeks
At 12 weeks
Normal control
33±0.4
35±0.4
34±0.3
Dietary induced untreated
36±0.2
180±2.2*
210±3.4*
Vardenafil 18mg./kg
38±0.6
175±2.4*
223±4.2*
Sildenafil 5mg./kg
35±0.3
169±3.2*
205±2.6*
* p<0.05
Table (6) : Multiple comparisons between different groups mean values of
serum TG level in ( mg/dl) using ANOVA test
Groups
Vardenafil
Sildenafil
Induced untreated
Normal control
–189*
–171*
Induced untreated
-13
5
Sildenafil
-18
–176*
* p<0.05
normal
dietary induced untreated
vardenafil
sildenafil
serum triglyceride in mg/dl
250
200
150
100
50
0
0 wk
6 wk
8 wk
10 wk
12 wk
Figure(2): Effect of vardenafil 18mg/kg/day and sildenafil 5mg./kg/day
treatment on serum TG level in comparison to the two control groups ( normal
3.3 Effect on HDL-C level
Similar serum HDL-C level was found in the zero time. This
level was significantly increased in groups (II, III, IV) fed with atherogenic
diet for 6 weeks( P<0.05). Remarkably it remained unchanged in normal
control group (I) as shown in table (7), and figure (3).
At the end of treatment (12 weeks), serum HDL-C level in groups (II, III,
IV) did not changed (P<0.05) as shown in table (7), and figure (3).
It was found that serum levels of HDL-C between the normal control
group (I) and the groups on high cholesterol diet (II,III &IV) significantly
differ, while The HDL-C values were not significantly different between the
untreated high cholesterol diet(II), vardenafil treated group (III) & sildenafil
treated group (IV), as shown in table (8).
Table (7): Changes of serum HDL-C level in mg/dl of the four experimental
groups.(N = 6 rabbits in each group). The values are expressed as mean
±SEM.
At zero time
At 6 weeks
At 12 weeks
Normal control
16.2±0.3
16.6±0.2
16.3±0.3
Dietary induced untreated
15.8±0.2
17.8±0.3*
19.1±0.2*
Vardenafil 18mg./kg
15.6±0.4
18.6±0.2*
18.5±0.4*
Sildenafil 5mg./kg
16.5±0.5
18.5±0.4*
19.6±0.4*
* p<0.05
Table (8) : Multiple comparisons between different groups mean values of
serum HDL-C level in ( mg/dl) using ANOVA test
Groups
Vardenafil
Sildenafil
Induced untreated
–2.2*
–3.3*
Induced untreated
0.6
-0.5
Sildenafil
1.1
Normal control
* p<0.05
-2.8*
normal
dietary induced untreated
vardenafil
sildenafil
20
serum HDL-C in mg/dl
17.5
15
12.5
10
zero time
6 wk
8 wk
10 wk
12 wk
Figure (3): Effect of Sildenafil 5mg/kg/day and Vardenafil 18mg./kg/day
treatment on serum HDL-C level in comparison to the two control group (normal
and induced untreated)
3.4 Effect on serum LDL-C level
At the zero time, the values of serum LDL-C were the same in all
groups of rabbits. Serum LDL-C level increased significantly in groups (II,
III, IV) fed with atherogenic diet for 6 weeks (P<0.05) as illustrated in table
(9) and figure (4).
For the another 6 weeks, serum LDL-C level goes increase in groups
(II, III, IV) fed with atherogenic diet(P<0.05) as shown in table (9) and
figure (4).
There was a statically significant difference (P<0.05) in serum levels
of LDL-C between the normal control group (I) and the groups on high
cholesterol diet (II,III &IV). While the LDL-C values did not change
significantly among the untreated high cholesterol diet(II), vardenafil treated
group (III) and sildenafil treated group (IV), as shown in table (10).
Table (9): Changes of serum LDL-C level in mg/dl of the four experimental
groups.(N = 6 rabbits in each group). The values are expressed as mean
±SEM.
At zero time
At 6 weeks
At 12 weeks
Normal control
25.2±1.7
26.6±2.25
26.5±1.5
Dietary induced untreated
23.3±2.26
418.0±4.5*
652.4±8.5*
Vardenafil 18mg./kg
22.9±1.6
459.0±4.9*
629.0±2.8*
Sildenafil 5mg./kg
23.5±1.9
444.7±4.5*
687.0±3.9*
* p<0.05
Table (10) : Multiple comparisons between different groups mean values of
serum LDL-C level in ( mg/dl) using ANOVA test
Groups
Vardenafil
Sildenafil
Induced untreated
Normal control
Induced untreated
Sildenafil
–602.5*
–660.5*
23.4
-34
–625.9*
58
* p<0.05
800
700
serum LDL-C mg/dl
600
500
400
300
200
100
0
zero time
Figure (4): Effect of
6 wk
8 wk
10 wk
12 wk
vardenafil 18mg/kg/day and sildenafil 5mg./kg/day
3.5 Effect on VLDL-C level
The initial values of serum VLDL-C were similar in all groups of
rabbits. After 6 weeks, serum levels of VLDL-C were significantly increased
in groups (II, III, IV) fed with atherogenic diet(P<0.05) and remained
unchanged in the normal control group (I) as shown in table (11), and figure
(5).
After 12 weeks on the high-cholesterol diet, serum levels of VLDL-C
further increased in groups (II, III, IV) (P<0.05). The changes in the levels
of serum VLDL-C in the four groups are summarized in table (11) and
figure (5).
There was a statistically significant difference(P<0.05) in serum levels
of VLDL-C between the normal control group (I) and the groups on high
cholesterol diet (II,III &IV). While the VLDL-C values were not
significantly different between the untreated high cholesterol diet(II) and
vardenafil treated group (III) and sildenafil treated group (IV), as shown in
table (12).
Table (11): Changes of serum VLDL-C level in mg/dl of the four
experimental group s.(N = 6 rabbits in each group). The values are
expressed as mean ±SEM.
At zero time
At 6 weeks
At 12 weeks
Normal control
6.6±0.08
7.0±0.08
6.8±0.06
Dietary induced untreated
7.2±0.04
36.0±0.44*
42.0±0.68*
Vardenafil 18mg./kg
7.6±0.12
35.0±0.48*
44.6±0.84*
Sildenafil 5mg./kg
7.0±0.06
33.8±0.64*
41.0±0.52*
* p<0.05
Table (12) : Multiple comparisons between different groups mean values of
VLDL-C level using ANOVA test
Groups
Vardenafil
Sildenafil
Induced untreated
–37.8*
–34.2*
Induced untreated
-2.6
1.0
Sildenafil
-3.6
Normal control
* p<0.05
–35.2*
normal
dietary induced untreated
vardenafil
sildenafil
50
45
serumVLDL-C inmg/dl
40
35
30
25
20
15
10
5
0
zero time
6 wk
8 wk
10 wk
12 wk
Figure (5): Effect of vardenafil 18mg/kg/day and sildenafil 5mg./kg/day
treatment on serum VLDL-C level in comparison to the two control groups
(normal and induced untreated)
3.6 Effect on atherogenic index
Initially, the atherogenic index values were similar in all groups of
rabbits. There was significant increase in atherogenic index (P<0.05) in
groups (II, III, IV) fed with atherogenic diet for 6 weeks as shown in table
(13), and figure (6).
After another 6 weeks, a statistically significant increase in
atherogenic index was noted in the induced untreated group, sildenafil
treated group and vardenafil treated group continued to have atherogenic
diet(P<0.05), as listed in table (13), and figure (6).
The Atherogenic index value of normal control group (I) was
statically significant differed (P<0.05) from the high cholesterol diet (II,III
&IV). While The Atherogenic index values were not significantly different
between the untreated high cholesterol diet(II), vardenafil treated group (III)
and sildenafil treated group (IV), as shown in table (14).
Table (13): Changes of atherogenic index in of the four experimental
groups.(N = 6 rabbits in each group). The values are expressed as mean
±SEM.
At zero time
At 6 weeks
At 12 weeks
Normal control
1.96±0.15
2.05±0.22
2.07±0.12
Dietary induced untreated
1.85±0.18
28.65±0.49*
44.51±0.99*
Vardenafil 18mg./kg
2.00±0.17
30.88±0.71*
41.07±0.23*
Sildenafil 5mg./kg
1.79±0.16
29.00±1.51*
43.35±0.17*
* p<0.05
Table (14) : Multiple comparisons between different groups mean values of
atherogenic index using ANOVA test
Groups
Vardenafil
Sildenafil
Induced untreated
–39.0*
–41.28*
Induced untreated
3.44
1.16
Sildenafil
2.28
Normal control
-42.44*
* p<0.05
normal
dietary induced untreated
vardenafil
sildenafil
50
45
atherogenic index
40
35
30
25
20
15
10
5
0
zero time
Figure (6): Effect of
6 wk
8 wk
10 wk
12 wk
vardenafil 18mg/kg/day and sildenafil 5mg./kg/day
3.7 Effect on serum level of MDA
Before the experiment, the baseline levels of serum MDA were not
significantly different among all groups. After 6 weeks of high cholesterol
diet, the MDA level significantly increased in induced untreated group (II)
(P<0.05) as well as sildenafil treated group (III) & vardenafil treated group
(IV) (P<0.05). After 12 weeks of high cholesterol diet, there was a further
significant increase in induced untreated (II) (P<0.05), while sildenafil
treated group (III) and vardenafil treated group (IV) were not significantly
different at (P<0.05), but it remained greater than the normal values. The
changes in MDA level are summarized in table (15) and figure (7).
The serum MDA level of sildenafil treated (III) and vardenafil treated
group (IV) was significantly (P<0.05) lower than that of induced untreated
group (II). There was no significant difference in MDA level between
sildenafil treated group (III) and vardenafil treated group (IV), as shown in
table (16)
Table (15): Sequential changes of serum MDA level mmole/L of the four experimental group. The
data expressed as Mean ±SEM (N=6 in each group) Using paired T-test.
At zero time
At 6 weeks
At 8 weeks
At 10 weeks
At 12 weeks
Normal control
2.1±0.10
2.4±0.20
2.6±0.10
2.5±0.21
2.4±0.20
Dietary induced untreated
2.5±0.20
3.4±0.30
3.9±0.40
4.5±0.43*
5.9±0.45*
Vardenafil 18mg./kg
2.6±0.30
3.5±0.41
3.7±0.35
3.9±0.30
4.1±0.22
Sildenafil 5mg./kg
2.4±0.25
3.3±.40
3.8±0.36
3.6±0.35
3.9±0.30
* p<0.05
Table (16): Multiple comparisons among different groups mean values of serum MDA level
mg/dl using ANOVA TEST
Groups
Vardenafil
Sildenafil
Induced untreated
Normal control
–1.7
–1.5
Induced untreated
1.8*
2.0*
Sildenafil
-0.2
* p<0.05
-3.5*
normal
dietary induced untreated
vardenafil
sildenafil
6
serum MDA level mmol/l
5
4
3
2
1
0
zero time
6 wk
8 wk
10 wk
12 wk
Figure (7) : the change in MDA Level in the four experimental group
3.8 Effect on serum reduced glutathione level
GSH
At zero time, the baseline levels of serum GSH were not significantly
different among all groups. After 6 weeks of high cholesterol diet, the GSH
level significantly decrease in induced untreated group (II) (P<0.05),
sildenafil treaded group (III) (P<0.05) & vardenafil treated group (IV)
(P<0.05). After 12 weeks of high cholesterol diet, there was a further
significant decrease in induced untreated (II) (P<0.05), while sildenafil treat
group (III) and vardenafil treated group (IV) not significantly different at
(P<0.05). The changes in GSH level are summarized in table (17) and figure
(8).
The serum GSH level of sildenafil treat group (III) and vardenafil
treated group (IV) was significantly (P<0.05) higher than that of induced
untreated group (II). There was no significant difference in GSH level
between sildenafil treat group (III)and vardenafil treated group (IV), as
shown in table (18)
Table (17): Sequential changes of serum GSH level uM of the four experimental groups. The data
expressed as Mean ±SEM (N=6 in each group) Using paired T-test.
At zero time
At 6 weeks
At 8 weeks
At 10 weeks
At 12 weeks
Normal control
60±2.2
59±2.4
62±2.1
65±2.2
58±2.1
Dietary induced untreated
62±2.4
45±2.1
39±1.8
33±1.7*
29±1.6*
Vardenafil 18mg./kg
58±2.0
42±2.0
39±1.9
37±1.8
40±1.8
Sildenafil 5mg./kg
63±2.5
44±2.1
40±2.0
38±2.0
39±1.7
* p<0.05
Table (18): Multiple comparisons among different groups mean values of serum
GSH level uM using ANOVA TEST
Groups
Vardenafil
Sildenafil
Induced untreated
Normal control
Induced untreated
Sildenafil
* p<0.05
18
19
-11*
-10*
-1
29*
normal
dietary induced untreated
vardenafil
sildenafil
70
serum GSH level in mmol/l
60
50
40
30
20
10
0
zero time
6 wk
8 wk
10 wk
12 wk
Figure (8): the change in Serum GSH Level in the four experimental groups
3.9 Effect on aortic intima-media thickness
(IMT)
At the end of 12 weeks of the study ,there was a statistically
significant increase (P<0.05) in aortic intima-media thickness (IMT) in the
induced untreated group, vardenafil treated group and sildenafil treated
group while remained unchanged in the Normal control group (I), as shown
in table (19) and figure (9).
The increase in IMT in vardenafil and sildenafil treated groups was found
to be lesser than that of induced untreated group, but there were no
statistically significant differences (P<0.05) in the means of IMT of
induced untreated group and treated groups (vardenafil and sidenafil
groups), as shown in table (20).
Table (19): Effect of atherogenic diet, Vardenafil 18mg/kg/day and Sildenafil
5mg/kg/day treatment on the rabbits aortic intima-media thickness measured in
(mm) (No. = 6 rabbits in each group).
At zero time
At 12 weeks
P-Value
Normal control
0.32±0.01
0.32±0.03
P>0.05
Dietary induced untreated
0.31±0.02
0.58±0.02
P<0.05
Vardenafil 18m/kg
0.30±0.022
0.56±0.02
P<0.05
Sildenafil 5mg/kg
0.34±0.021
0.57±0.01
P<0.05
Table (20) : Multiple comparisons between different groups mean values of
rabbits aortic intima-media thickness in measured in (mm) by using ANOVA test
Groups
Vardenafil
Sildenafil
Induced untreated
-0.24*
-0.25*
- 0.26*
Induced untreated
0.02
0.01
Sildenafil
0.01
Normal control
*P<0.05
zero time
12 weeks
aortic intima-media thickness
0.6
0.5
0.4
0.3
0.2
0.1
0
normal
dietary induced
untreated
ardenafil
sildenafil
Figure (9): Effect of Vardenafil 18mg/kg/day and Sildenafil 5mg/kg/day treatment on rabbits
aortic intima-media thickness (IMT) measured in (mm) in comparisons to the two control group
(normal and induced untreated)
3.10 Effect on aortic diameter
At the end of 12 weeks of the study ,there was a statistically significant
increase (P<0.05) in aortic diameter in the induced untreated group,
vardenafil treated group and sidenafil treated group and remained
unchanged in the normal control group (I), as shown in table (21) and figure
(10).
The increase in aortic diameter in vardenafil and sidenafil treated groups
was found to be close to that of induced untreated group, but there was no
statistically significant differences (P<0.05) in the means of aortic diameter
of induced untreated group and treated groups (vardenafil and sildenafil
groups),as shown in table (22).
Table (21): Effect of atherogenic diet, vardenafil 18mg/kg/day and
sildenafil5mg/kg/day treatment on rabbits aortic diameter measured in
(mm) (No. = 6 rabbits in each group).
At zero time
At 12 weeks
P-Value
Normal control
2.10 ± 0.08
2.28 ± 0.07
p>0.05
Dietary induced untreated
2.20± 0.17
2.70 ± 0.04
P<0.05
Vardenafil 18m/kg
2.17 ± 0.17
2.65 ± 0.07
P<0.05
Sildenafil 5mg/kg
2.11 ± 0.15
2.59 ± 0.05
P<0.05
Table (22): Multiple comparisons between different groups mean values of
rabbits aortic diameter measured in (mm) by using ANOVA test
Groups
Vardenafil
Sildenafil
Induced untreated
Normal control
-0.37*
-0.31*
Induced untreated
0.05
0.11
Sildenafil
-0.06
- 0.42*
*P<0.05
zero time
12 weeks
aortuc diameter in mm
3
2.5
2
1.5
1
0.5
0
normal
dietary induced
untreated
vardenafil
sildenafil
Figure (10): Effect of vardenafil 18mg/kg/day and sildenafil 5mg/kg/day
treatment on rabbits aortic diameter measured in (mm) in comparisons to the
3.11 Effect on aortic peak systolic blood flow
velocity (PSV)
At the end of 12 weeks of the study, there was an increase in aortic PSV
in the induced untreated group and sidenafil treated group and there was a
decrease in aortic PSV in vardenafil treated group and remained unchanged
in the normal control group. But all these changes were statistically not
significant (P>0.05), as shown in table(23) and figure (11).
There was no statistically significant differences (P>0.05) in the means of
aortic PSV of different groups as shown in table (24).
Table (23): Effect of atherogenic diet, vardenafil 18mg/kg/day and sildenafil
5mg/kg/day treatment on the rabbits aortic peak systolic blood flow velocity
measured in (cm/s) (No. = 6 rabbits in each group).
At zero time
At 12 weeks
P-Value
Normal control
29.0±2.1
31.1±2.3
p>0.05
Dietary induced untreated
30.0±2.4
32.0±3.0
p>0.05
Vardenafil 18m/kg
30.5±3.1
28.5±3.2
p>0.05
Sildenafil5mg/kg
30.2±2.9
31.4±2.6
p>0.05
Table (24): Multiple comparisons between different groups mean values of rabbits
aortic peak systolic blood flow velocity measured in (cm/s) by using ANOVA test
Groups
Vardenafil
Sildenafil
Induced untreated
Normal control
2.6
-0.3
Induced untreated
3.5
0.6
Sildenafil
2.9
- 0.9
aortic PSV in cm/sec
zero time
12 week
39.5
38
36.5
35
33.5
32
30.5
29
27.5
26
24.5
23
21.5
20
normal
dietary induced
untreated
vardenafil
sildenafil
Figure (11): Effect of vardenafil 18mg/kg/day and sildenafil 5mg/kg/day
treatment on rabbits aortic peak systolic velocity (PSV) measured in (cm/s) in
comparisons to the two control group ( normal and induced untreated)
3.12 Effect on aortic end diastolic blood flow
velocity (EDV)
At the end of 12 weeks of the study, there was a increase in aortic EDV
in the induced untreated group and there was a decrease in aortic EDV in
the vardenafil treated group, and sildenafil treated group. But all these
changes were statistically not significant (P>0.05) as shown in table (25) and
figure (12).
There was no statistically significant difference (P>0.05) in the means of
aortic EDV of different groups, as shown in table (26).
Table (25): Effect of atherogenic diet, vardenafil 18mg/kg/day and
sildenafil 5mg/kg/day treatment on the rabbits aortic end diastolic blood
flow velocity measured in (cm/s) (No. = 6 rabbits in each group).
At zero time
At 12 weeks
P-Value
Normal control
4.2 ±0.56
4.7±0.62
p>0.05
Dietary induced untreated
4.6 ±1.2
5.2±0.36
p>0.05
Vardenafil 18m/kg
5.2 ±0.8
4.9±0.54
p>0.05
Sildenafil 5mg/kg
3.9 ±0.48
3.75±0.68
p>0.05
Table (26): Multiple comparisons between different groups mean values of
rabbits aortic end diastolic blood flow velocity measured in (cm/s) by using
ANOVA test
Groups
Vardenafil
Sildenafil
Induced untreated
Normal control
-0.2
0.95
-0.5
Induced untreated
0.3
1.45
Sildenafil
-1.15
Zero time
12 week
aortic EDV in cm/sec
6
5
4
3
2
1
0
Normal
Dietary
Induced
Untreated
Vardenafil
Sildenafil
3.13 Effect on aortic blood flow resistive index
(RI)
At the end of 12 weeks of the study, there was a significant increase
(P>0.05) in aortic blood flow RI in the induced untreated group, sildenafil
treated group and vardenafil treated group, and remained unchanged in the
normal control group, as shown in table (27) and figure (13).
There was only statistically significant difference (P<0.05) between the
means of aortic RI of normal control group and induced untreated group,
sildenafil treated group and vardenafil treated group. No statistically
significant differences (P>0.05) were observed between means of aortic RI
of induced untreated group, sildenafil treated group and vardenafil treated
group, as shown in table (28).
Table (27): Effect of atherogenic diet, vardenafil 18mg/kg/day and sildenafil
5mg/kg/day treatment on the rabbits aortic blood flow resistive index (No. = 6
rabbits in each group).
At zero time
At 12 weeks
P-Value
Normal control
0.82±0.019
0.84±0.02
p>0.05
Dietary induced untreated
0.83±0.022
0.91±0.021
P<0.05
Vardenafil 18m/kg
0.83±0.034
0.9±0.021
P<0.05
Sildenafil5mg/kg
0.85±0.026
0.89±0.031
P<0.05
Table (28): Multiple comparisons between different groups mean values of
rabbits aortic blood flow resistive index by using ANOVA test
Groups
Vardenafil
Sildenafil
Induced untreated
Normal control
-0.06*
-0.05*
Induced untreated
0.01
0.02
Sildenafil
-0.01
*P<0.05
- 0.07*
zero time
0.92
12 weeks
Aortic blood flow RI
0.9
0.88
0.86
0.84
0.82
0.8
0.78
0.76
normal
dietary induced
untreated
vardenafil
sildenafil
Figure (13): Effect of vardenafil 18mg/kg/day and sildenafil 5mg/kg/day
treatment on rabbits aortic blood flow resistive index (RI) in comparisons to the
two control group (normal and induced untreated)
3.14 Effect on aortic blood flow pulsatality
index (PI)
At the end of 12 weeks of the study, there was a significant increase
(P>0.05) in aortic blood flow PI in the induced untreated group, sildenafil
treated group and vardenafil treated group, and remained unchanged in the
normal control group, as shown in table (29) and figure (14).
There was only statistically significant difference (P<0.05) between the
means of aortic PI of normal control group and induced untreated group,
sildenafil treated group and vardenafil treated group. No statistically
significant differences (P>0.05) were observed between means of aortic PI
of induced untreated group, sildenafil treated group and vardenafil treated
group, as shown in table (30).
Table (29): Effect of atherogenic diet, vardenafil 18mg/kg/day and sildenafil
5mg/kg/day treatment on the rabbits aortic blood flow pulsatality index (No. = 6
rabbits in each group).
At zero time
At 12 weeks
P-Value
1.95±0.1
1.83±0.11
p>0.05
Dietary induced untreated
1.88±0.097
2.9±0.079
P<0.05
Vardenafil 18m/kg
1.9±0.093
2.77±0.15
P<0.05
Sildenafil5mg/kg
2.01±0.16
2.85±0.11
P<0.05
Normal control
Table (30): Multiple comparisons between different groups mean values of
rabbits aortic blood flow pulsatality index by using ANOVA test
Groups
Vardenafil
Sildenafil
Induced untreated
-0.94*
-1.02*
- 1.07*
Induced untreated
0.31
0.05
Sildenafil
0.08
Normal control
*P<0.05
zero time
12 weeks
3
Aortic flow PI
2.5
2
1.5
1
0.5
0
normal
dietary induced
untreated
vardenafil
sildenafil
Figure (14): Effect of vardenafil 18mg/kg/day and sildenafil 5mg/kg/day
treatment on rabbits aortic blood flow pulsatality index (PI) in comparisons to
3.15 Effect on renal artery peak systolic blood
flow velocity (PSV)
At the end of 12 weeks of the study, there was statistically significant
increase (P<0.05) in renal artery PSV in the induced untreated group, but no
statistically significant changes (P>0.05) were observed in other groups, as
shown in table (31) and figure (15).
There was statistically significant difference (P<0.05) between the
means of renal artery PSV of the induced untreated group and other three
groups, as shown in table (32).
Table (31): Effect of atherogenic diet, vardenafil 18mg/kg/day and sildenafil
5mg/kg/day treatment on the rabbits renal artery peak systolic velocity
measured in cm/s (No. = 6 rabbits in each group).
At zero time
At 12 weeks
P-Value
Normal control
45.2±4.6
46.1±5.6
p>0.05
Dietary induced untreated
46.1±3.4
69.0±8.5
P<0.05
Vardenafil 18m/kg
44.5±4.1
58.0±4.5
p>0.05
Sildenafil 5mg/kg
46.0±4.2
57.0±3.5
p>0.05
Table (32): Multiple comparisons between different groups mean values of
rabbits renal artery peak systolic blood flow velocity measured in cm/s by using
ANOVA test .
Groups
Vardenafil
Sildenafil
Induced untreated
Normal control
-11.9
-10.9
Induced untreated
11*
12*
Sildenafil
*P<0.05
-1
- 22.9*
zero time
12weeks
70
renal artery PSV cm/sec
60
50
40
30
20
10
0
normal
dietary induced
untreated
vardenafil
sildenafil
Figure (15): Effect of vardenafil 18mg/kg/day and sildenafil 5mg/kg/day
treatment on rabbits renal artery peak systolic velocity (PSV) measured in cm/s
in comparisons to the two control group (normal and induced untreated)
3.16 Effect on renal artery end diastolic blood
flow velocity (EDV)
At the end of 12 weeks of the study, there was a statistically
significant decrease (P<0.05) in renal artery EDV in the vardenafil and
sildenafil treated groups, but no statistically significant changes (P>0.05)
were observed in normal group and induced untreated group, as in table
(33) and figure (16).
There was no statistically significant difference (P<0.05) between the
means of renal artery EDV of the vardenafil treated group and sildenafil
treated group, as shown in table (34).
Table (33): Effect of atherogenic diet, , vardenafil 18mg/kg/day and sildenafil
5mg/kg/day treatment on the rabbits renal artery end diastolic velocity measured
in cm/s (No. = 6 rabbits in each group).
At zero time
At 12 weeks
P-Value
Normal control
23.0±2.4
24.1±2.8
p>0.05
Dietary induced untreated
22.6±2.3
23.3±4.0
p>0.05
Vardenafil 18m/kg
23.9±2.1
18.2±1.8
P<0.05
Sildenafil5mg/kg
24.6±1.8
19.0±1.2
P<0.05
Table (34): Multiple comparisons between different groups mean values of
rabbits renal artery end diastolic blood flow velocity measured in cm/s by using
ANOVA test
Groups
Vardenafil
Sildenafil
Induced untreated
Normal control
5.9*
5.1*
Induced untreated
5.1*
4.3*
Sildenafil
0.8
0.8
*P<0.05
zerc time
12weeks
renal artery EDV in cm/sec
25
20
15
10
5
0
normal
dietary induced
untreated
vardenafil
sildenafil
Figure (16): Effect of vardenafil 18mg/kg/day and sildenafil 5mg/kg/day
treatment on rabbits renal artery end diastolic velocity (EDV) measured in cm/s
3.17 Effect on renal artery blood flow resistive
index (RI)
At the end of 12 weeks of the study ,there was a statistically significant
increase (P<0.05) in renal artery blood flow RI only in the induced
untreated group. No statistically significant changes (P>0.05) were observed
in other groups, as shown in table (35) and figure (17).
There was a statistically significant difference (P<0.05) between the
means of renal artery RI of the induced untreated group and other three
groups, as shown in table (36).
Table (35): Effect of atherogenic diet, , vardenafil 18mg/kg/day and sildenafil
5mg/kg/day treatment on the rabbits renal artery blood flow resistive index (No.
= 6 rabbits in each group).
At zero time
At 12 weeks
P-Value
Normal control
0.478 ± 0.024
0.480 ± 0.01
p>0.05
Dietary induced untreated
0.461± 0.013
0.647 ± 0.043
P<0.05
Vardenafil 18m/kg
0.482 ± 0.021
0.522 ± 0.025
p>0.05
Sildenafil 5mg/kg
0.496 ± 0.022
0.539 ± 0.027
p>0.05
Table (36): Multiple comparisons between different groups mean values of
rabbits renal artery blood flow resistive index by using ANOVA test
Groups
Vardenafil
Sildenafil
Induced untreated
Normal control
-0.042
-0.059
- 0.167*
Induced untreated
0.125*
0.108*
Sildenafil
0.017
*P<0.05
zero time
12 weeks
0.7
Renal artery RI
0.6
0.5
0.4
0.3
0.2
0.1
0
normal
dietary induced
untreated
vardenafil
sildenafil
Figure (17): Effect of vardenafil 18mg/kg/day and sildenafil 5mg/kg/day
treatment on rabbits renal artery blood flow resistive index (RI) in comparisons
to the two control group (normal and induced untreated)
3.18 Effect on renal
pulsatality index (PI)
artery
blood
flow
At the end of 12 weeks of the study, there was statistically significant
increase (P<0.05) in renal artery blood flow PI only in the induced untreated
group. No statistically significant changes (P>0.05) were observed in other
groups, as shown in table (37) and figure (18).
There was statistically significant difference (P<0.05) between the means
of renal artery PI of induced untreated group and other three groups, as
shown in table (38).
Table (37): Effect of atherogenic diet, , vardenafil 18mg/kg/day and sildenafil
5mg/kg/day treatment on the rabbits renal artery blood flow pulsatality index
(No. = 6 rabbits in each group).
At zero time
At 12 weeks
P-Value
Normal control
0.74±0.057
0.72±0.021
p>0.05
Dietary induced untreated
0.68±0.026
1.12±0.13
P<0.05
Vardenafil 18m/kg
0.79±0.055
0.837±0.06
p>0.05
Sildenafil 5mg/kg
0.76±0.054
0.85±0.059
p>0.05
Table (38): Multiple comparisons between different groups mean values of
rabbits renal artery blood flow pulsatality index by using ANOVA test
Groups
Vardenafil
Sildenafil
Induced untreated
Normal control
-0.117
-0.13
- 0.4*
Induced untreated
0.283*
0.27*
Sildenafil
-0.013
*P<0.05
zero time
12 weeks
1.2
Renal artery PI
1
0.8
0.6
0.4
0.2
0
normal
dietary induced
untreated
vardenafil
sildenafil
3.19 Effect on intra-renal arteries peak systolic
blood flow velocity (PSV)
At the end of 12 weeks of the study, there was statistically significant
increase (P<0.05) in intra-renal arteries PSV in the induced untreated group,
but no statistically significant changes (P>0.05) were observed in other
groups, as shown in table (39) and figure (19).
There was statistically significant difference (P<0.05) between the means
of renal artery PSV of the induced untreated group and other three groups,
as shown in table (40).
Table (39): Effect of atherogenic diet, , vardenafil 18mg/kg/day and sildenafil
5mg/kg/day treatment on the rabbits intra- renal arteries peak systolic velocity
measured in cm/s (No. = 6 rabbits in each group).
At zero time
At 12 weeks
P-Value
Normal control
41.6±6.7
42.4±6.6
p>0.05
Dietary induced untreated
41.2±±3.4
64.1±7.8
P<0.05
Vardenafil 18m/kg
42.2±5.5
48.3±4.2
p>0.05
Sildenafil 5mg/kg
40.7±2.7
49.1±2.3
p>0.05
Table (40): Multiple comparisons between different groups mean values of the
rabbits intra- renal arteries peak systolic velocity measured in cm/s by using
covariance ANOVA test
Groups
Vardenafil
Sildenafil
Induced untreated
Normal control
Induced untreated
Sildenafil
*P<0.05
-5.9
-6.7
15.8*
15*
0.8
- 21.7*
Intra-renal artery PSV in cm/sec
zero time
12 weeks
70
60
50
40
30
20
10
0
normal
dirtary induced
untreated
vardenafil
sildenafil
Figure (19): Effect of vardenafil 18mg/kg/day and sildenafil 5mg/kg/day
treatment on the rabbits intra- renal arteries peak systolic velocity (PSV)
measured in cm/s in comparisons to the two control group (normal and induced
untreated)
3.20 Effect on intra-renal arteries end diastolic
blood flow velocity (EDV)
At the end of 12 weeks of the study, there was statistically significant
decrease (P<0.05) in intra-renal arteries EDV in the vardenafil and
sildenafil treated groups, but no statistically significant changes (P>0.05)
were observed in normal group and induced untreated group, as shown in
table (41) and figure (20).
There was no statistically significant difference (P<0.05) between the
means of intra-renal arteries EDV of the vardenafil treated group and
sildenafil treated group, as shown in table (42).
Table (41): Effect of atherogenic diet, vardenafil 18mg/kg/day and sildenafil
5mg/kg/day treatment on the rabbits intra- renal arteries end diastolic velocity
measured in cm/s (No. = 6 rabbits in each group).
At zero time
At 12 weeks
P-Value
Normal control
22 ± 0.62
23.1 ±0.51
p>0.05
Dietary induced untreated
22.3 ±0.36
24.2 ±0.43
p>0.05
Vardenafil 18m/kg
22.4 ±0.41
19 ±0.32
P<0.05
Sildenafil 5mg/kg
21.9 ±0.32
18.4 ±0.37
P<0.05
Table (42): Multiple comparisons between different groups mean values of
rabbits intra- renal arteries end diastolic velocity measured in cm/s by using
ANOVA test
Groups
Vardenafil
Sildenafil
Induced untreated
Normal control
4.1*
4.7*
-1.1
Induced untreated
5.2*
5.8*
Sildenafil
-0.6
*P<0.05
intra-renal artery EDV in cm/sec
zero time
12weeks
25
20
15
10
5
0
normal
dietary induced
untreated
vardenafil
sildenafil
3.21 Effect on intra-renal arteries blood flow
resistive index (RI)
At the end of 12 weeks of the study ,there was statistically significant
increase (P<0.05) in intra-renal arteries blood flow RI only in the induced
untreated group. No statistically significant changes (P>0.05) were observed
in other groups, as shown in table (43) and figure (21).
There was statistically significant difference (P<0.05) between the
means of intra-renal arteries RI of induced untreated group and other three
groups, as shown in table (44).
Table (43): Effect of atherogenic diet, vardenafil 18mg/kg/day and sildenafil
5mg/kg/day treatment on the rabbits intra-renal arteries blood flow resistive
index (No. = 6 rabbits in each group).
At zero time
At 12 weeks
P-Value
Normal control
0.461 ± 0.022
0.462 ±0.018
p>0.05
Dietary induced untreated
0.472±0.016
0.63±0.031
P<0.05
Vardenafil 18m/kg
0.46±0.024
0.51 ±0.038
p>0.05
Sildenafil 5mg/kg
0.48 ±0.029
0.53 ±0.021
p>0.05
Table (44): Multiple comparisons between different groups mean values of
rabbits intra-renal arteries blood flow resistive index by using ANOVA test
Groups
Vardenafil
Sildenafil
Induced untreated
Normal control
-0.048
-0.068
Induced untreated
0.12*
0.1*
Sildenafil
-0.02
*P<0.05
- 0.168*
zero time
12 weeks
0.7
Intr-renal artery RI
0.6
0.5
0.4
0.3
0.2
0.1
0
normal
dietary induced
untreated
vardenafil
sildenafil
Figure (21): Effect of vardenafil 18mg/kg/day and sildenafil 5mg/kg/day
treatment on rabbits intra-renal arteries blood flow resistive index (RI) in
comparisons to the two control group (normal and induced untreated)
3.22 Effect on intra-renal arteries blood flow
pulsatality index (PI)
At the end of 12 weeks of the study, there was statistically significant
increase (P<0.05) in intra-renal arteries blood flow PI only in the induced
untreated group. No statistically significant changes (P>0.05) were observed
in other groups, as shown in table (45) and figure (22).
There was statistically significant difference (P<0.05) between the means
of intra-renal arteries PI of induced untreated group and other three groups,
as shown in table (46).
Table (45): Effect of atherogenic diet, , vardenafil 18mg/kg/day and sildenafil
5mg/kg/day treatment on the rabbits intra-renal arteries blood flow pulsatality
index (No. = 6 rabbits in each group).
At zero time
At 12 weeks
P-Value
Normal control
0.75± 0.051
0.73±0.043
P>0.05
Dietary induced untreated
0.71±0.035
1.1±0.084
P<0.05
Vardenafil 18m/kg
0.78±0.055
0.83±0.083
P>0.05
Sildenafil 5mg/kg
0.77±0.063
0.86±0.044
P>0.05
Table (46): Multiple comparisons between different groups mean values of
rabbits intra-renal arteries blood flow pulsatality index by using ANOVA test
Groups
Vardenafil
Sildenafil
Induced untreated
Normal control
-0. 1
-0.13
- 0.37*
Induced untreated
0.27*
0.24*
Sildenafil
0.03
*P<0.05
zero time
1.2
12 weeks
Intra-renal artery PI
1
0.8
0.6
0.4
0.2
0
normal
dietary induced
untreated
Figure (22): Effect of
vardenafil
sildenafil
vardenafil 18mg/kg/day and sildenafil 5mg/kg/day
3.23 Effect on renal artery-to-aortic
systolic velocity ratio (RAR)
peak
At the end of 12 weeks of the study ,there was statistically significant
increase (P<0.05) in (RAR) in the induced untreated group, vardenafil
treated group and sidenafil treated group and remained unchanged in the
normal control group (I), as shown in table (47) and figure (23).
The increase of (RAR) in vardenafil and sidenafil treated groups was
found to be lesser than that of induced untreated group, but there were no
statistically significant differences (P<0.05) in the means of (RAR) of
induced untreated group and treated groups (vardenafil and sildenafil
groups), as shown in table (48).
Table (47): Effect of atherogenic diet, , vardenafil 18mg/kg/day and sildenafil
5mg/kg/day treatment on rabbits renal artery-to-aortic peak systolic velocity
ratio (No. = 6 rabbits in each group).
At zero time
At 12 weeks
P-Value
Normal control
2.00 ± 0.24
2.19 ±0.25
p>0.05
Dietary induced untreated
1.99 ±0.23
2.78 ±0.17
P<0.05
Vardenafil 18m/kg
2.01 ±0.27
2.65 ±0.23
P<0.05
Sildenafil 5mg/kg
1.98 ±0.24
2.72 ±0.36
P<0.05
Table (48): Multiple comparisons between different groups mean values of
rabbits renal artery-to-aortic peak systolic velocity ratio by using ANOVA test
Groups
Vardenafil
Sildenafil
Induced untreated
Normal control
-0. 46*
-0.53*
Induced untreated
0.13
0.06
Sildenafil
0.07
*P<0.05
- 0.59*
zero time
12 weeks
Renal artery/Aortic ratio
3
2.5
2
1.5
1
0.5
0
normal
dietary induced untreated
vardenafil
sildenafil
Figure (23): Effect of vardenafil 18mg/kg/day and sildenafil 5mg/kg/day
treatment on rabbits renal artery-to-aortic peak systolic velocity ratio in
comparisons to the two control group (normal and induced untreated)
3.24 Histopathological findings
At the end of the study a cross section of normal rabbits aorta shows
the normal appearance of all three arterial wall layers (intima, media and
adventitia). The intima-media lining shows no thickening in all rabbits of
this group (100%). There was statistically significant difference between
normal control group and other three groups (P<0.05), as shown in table (49)
& (50) and figure (24) & (25). While High cholesterol diet untreated had
virtually a 100% involvement of aorta with different severity of
atherosclerosis (all animals had a degree of atherosclerosis). 16.7% of the
group had initial atherosclerotic lesion as a phase II, 33.33% of the group
had intermediate atherosclerotic lesion as phase III and 50% of the group
had advance atherosclerotic lesion (33.33%as phase IV, and 16.7% as phase
V) . There was statistically significant difference between induced untreated
group and normal control group (P<0.05), as shown in table (49) & (50) and
figure (24) & (25). Together sildenafil treated group results in non
significant reduction in severity of atherosclerotic lesion compared with
induced untreated group. 16.7 % of rabbits treated with sildenafil had initial
atherosclerotic lesion as a phase II, 50% of the group had intermediate
atherosclerotic lesion appear as phase III, and 33.33% of the group had
advance atherosclerotic lesion as phase IV. There was no complicated lesion
in sildenafil treated group, as shown in table (49) & (50) and figure (24) &
(25).In concert vardenafil treated group results in non significant reduction
in severity of atherosclerotic lesion compared with induced untreated group.
33.33 % of rabbits treated with vardenafil had initial atherosclerotic lesion
as a phase II, 33.33% of the group had intermediate atherosclerotic lesion
appear as phase III, and 33.33% of the group had advance atherosclerotic
lesion as phase IV. There was no complicated lesion in vardenafil treated
group, as shown in table (49) & (50) and figure (24) & (25).
Table (49): Demonstration of different aortic atherosclerotic phases for different
rabbit groups at the end of 12 weeks of the study
Phases of atherosclerotic lesion
groups
Normal
Phase 1
Phase 2
Phase 3
NO.
6
0
0
0
%
100
0
0
NO.
0
0
%
0
Sildenafil
NO.
5mg/kg/day
Vardenafil
Normal control
NO.= 6
Dietary induced
untreated
Phase 4
Phase 5
Phase 6
0
0
0
0
0
0
0
1
2
2
1
0
0
16.7
33.33
33.33
16.7
0
0
0
1
3
0
0
%
0
0
16.7
50
33.33
0
0
NO.
0
0
2
2
2
0
0
%
0
0
33.33
33.33
33.33
0
0
NO.= 6
18mg/kg/day
2
120
standard diet
high cholest diet
sildenafil
vardenafil
Percentage of involvment
100
80
60
40
20
0
normal
phase 1
phase 2
phase 3
phase 4
phase 5
Figure (24): Percentages of aortic involvement with different atherosclerotic
phases.
Table (50): Demonstration of different aortic atherosclerotic lesions for different
rabbit groups at the end of 12 weeks of the study
Normal
Normal control
NO= 6
NO.
Initial lesion
Intermediate
lesion
Advance
lesion
Complicated
lesion
6
0
0
0
0
%
100
0
0
0
0
Dietary induced
untreated
NO= 6
NO.
0
1
2
2
1
%
0
16.7
33.33
33.33
16.7
Sildenafil
NO.
0
1
3
2
0
5mg/kg/day
%
0
16.7
50
33.33
0
Vardenafil
NO.
0
2
2
0
18mg/kg/day
%
0
33.33
33.33
0
2
33.33
120
stand diet
high cholest diet
sildenafil
vardenafil
Percentage of involvment
100
80
60
40
20
0
Normal
Initia
Intermedia
Advance
Complicat
Figure (25): Percentages of aortic involvement with different atherosclerotic lesions.
Lumen
Adventitia
Figure (26): A cross section of normal rabbit aorta. The section stained with
haematoxylin and eosin (×10)
Foam cells
Lumen
Figure(27): A cross section of hypercholesterolemic rabbit's aorta. The section
stained with haematoxylin and eosin ( ×10)
Layer of foam cells
Figure (28) A cross section of hypercholesterolemic rabbit's aorta. The section
stained with haematoxylin and eosin ( ×10)
Intimal thickness
Figure (29):A cross section from aorta shows a pathological intimal thickening. The
section stained with haematoxylin and eosin ( ×10)
Preatheroma lesion
Absence of fatty core
Figure (30):A cross section from aorta shows a pathological intimal thickening. The
section stained with haematoxylin and eosin ( ×10)
Foam cell
Intimal thickness
Figure (31): A cross section of hypercholesterolemic rabbit's aorta. The section
stained with haematoxylin and eosin ( ×40)
Thin layer of fibrous cap
Necrotic fatty core
Figure (32): A cross section from aorta shows a fibro-atheromatous plaque. The
section stained with haematoxylin and eosin ( ×10)
Figure (33): A B-mode ultrasound image showing a rabbit abdominal aorta with
normal aortic wall thickness , D1 is the intima-media thickness = 0.3 mm , D2 is the
aortic diameter = 2.1 mm
Figure (34):A B-mode ultrasound image showing a rabbit abdominal aorta with an
increased aortic thickness (sclerotic wall), but there is no apparent plaques , D1 is
the intima-media thickness = 0.6 mm , D2 is the aortic diameter = 2.8 mm
Plaque
Figure (35): A B-mode ultrasound image showing a rabbit abdominal aorta with an
increased aortic thickness (sclerotic wall), there also dissemination of plaques inside
the aorta , D1 is the intima media thickness = 0.7 mm
Figure (36): A Doppler wave form spectrum showing a rabbits aortic blood flow
velocity, PSV=0.49 m/s , EDV=0.06 m/s RI=0.88 , S/D ratio=8.2
Figure (37): A Doppler wave form spectrum showing a rabbits renal artery blood
flow velocity, PSV=0.48 m/s , EDV=0.17m/s, RI=0.58 , S/D ratio=2.4
Figure (38): A Doppler wave form spectrum showing a rabbits intra-renal artery
blood flow velocity, PSV=0.49 m/s , EDV=0.20m/s, RI=0.59 , S/D ratio=2.5
4.1 Effect on Serum Lipid Profile
4.1.1 Effect of cholesterol enriched diet on serum lipid profile and
atherogenic index
The cholesterol-fed rabbit is a model frequently used for studying the
development and modifications in the natural history of atherosclerosis(174).
In the current study, feeding of atherogenic diet to rabbits for 12
weeks resulted in marked hypercholesterolemia in which serum TC, TG,
LDL-C, HDL-C, VLDL and atherogenic index levels were found to be
increased in agreement with changes in these parameters which have been
reported earlier Romero et al. (2000)(175) ; Prasad et al, ( 2007)(176) and
Nigris et al. (2008)(177) and these results might be attributed to exogenous
cholesterol (atherogenic diet).
4.1.2 Effect of Sildenafil and Vardenafil treatment on serum lipid
profile and atherogenic index
In this study, there was insignificant changes in serum TC, TG, HDL,
LDL, VLDL and atherogenic index levels (p<0.05) in sildenafil and
vardenafil treated rabbits as compared with that in the induced untreated
group. This may be due to that the change in lipid parameter induced by
high cholesterol diet (2% cholesterol for 12 weeks) overrides any changes
from sildenafil and vardenafil treatment. However Kimura et al. (2003)
found no significant differences in lipid parameter in smokers treated with
sildenafil(178).
4.2 Effect on oxidative stress
4.2.1 Effect of high cholesterol diet on oxidative stress
In the present study, hypercholesterolemic atherosclerosis was
associated with increases in the serum levels of the lipid peroxidation
product MDA, and decrease in the level of GSH suggesting an increase in
the levels or activity of oxygen radicals. MDA and GSH have been
considered as specific indicators of oxidative status (160). Increases in serum
MDA and a decrease in GSH in hypercholesterolemic rabbit were also
observed by Jale et al. (2004)(179).
Moreover, increases in the levels of serum MDA in high cholesterolfed rabbits have been reported previously (180,181). Hypercholesterolemia
could increase levels of reactive oxygen species in various ways.
Hypercholesterolemia increases cholesterol content of platelets,
polymorphonuclear leukocytes (PMNs) and endothelial cells. This result
would lead to increase in activated complements 3 and 5 (C3a and C5a),
platelet activating factor, interleukin-1, and tumor necrosis factor, and
synthesis of prostaglandins and leukotriene (182,183,184).
Thus our study further confirms that the increase in oxidative stress
and subsequent lipid peroxidation together with the decrease in the level of
antioxidant may provide mechanistic answer for atherosclerosis induced by
high fat diet in rabbit.
4.2.2 Effect of Sildenafil and Vardenafil on MDA and GSH level
Sildenafil and vardenafil had significant effect on serum MDA and
GSH levels. Hence it inhibited the increase of serum MDA induced in high
cholesterol-fed rabbits suggesting decrease in ROS and subsequent lipid
peroxidation. Also these drugs prevent GSH depletion in
hypercholesterolemia rabbit, and thus, maintain antioxidant reserve which is
crucial for vascular protection against lipid peroxidation. This finding is
consistent with Abdollahi et al. (2003)(185) who found that the use of cAMP
and cGMP phosodiesterase inhibitors, theophylline and sildenafil, prevented
lead induced increased lipid peroxidation and also protected from decreased
thiol groups content and total antioxidant power of the gland and secretions.
The same trend of effects was observed in gland and saliva. The antioxidant
activity of PDEIs can be explained by that first. It is increase total antioxidant capacity by increasing the activities of CAT, GSH-Px, and total
SOD(186). Second, sildenafil increase nitric oxide which activates guanylate
cyclase resulting in an increase of the conversion of GTP to cGMP and
cGMP which mediates an anti-oxidant action(185). Third, sildenafil could
reduce NAD(P)H oxidase expression and superoxide formation in corpus
cavernosum of hypercholesterolemic rabbits (186).
4.3 Effect on aortic intima-media thickness
4.3.1 Effect of cholesterol enriched diet on aortic intima-media thickness
A significant increase in aortic intima-media thickness (P<0.05) was
found in rabbits fed with cholesterol enriched diet as compared with that of
the normal control group. This result is in agreement with that reported by
Michael et al. (1999)(187) and Roberto et al. (2004)(188) who examined
rabbits by high-resolution magnetic resonance imaging (MRI) and found that
the vessel wall thickness increased significantly in cholesterol fed rabbits.
the increment in aortic intima-media thickness might be due to lipid
deposition on aortic wall.
4.3.2 Effect of Sildenafil and Vardenafil treatment on aortic intimamedia thickness
There was insignificant reduction of aortic intima-media thickness in
sildenafil and vardenafil treated rabbits as compared with that in the induced
untreated group. Although sildenafil and vardenafil have the ability to
improve endothelial function, and possess antioxidant action. Additionally
sildenafil reduces endothelial proliferation as mentioned by, (Heller et al.)
who concluded that the antiproliferative effect of nitric oxide was
independent of cGMP, which would exclude a cGMP-dependent mechanism
involving the protein kinase G(189).
This finding may be due to the fact that rabbits were continued to be
fed with cholesterol enriched diet together with the treatment with
sildenafil and verdenafil which may prevent the maximal effect of the
drugs or due to low doses of drugs. Another reason for the non significant
reduction in aortic intima-media thickness may be due to small sample size
studied and short duration of treatment.
4.4 Effect on aortic diameter
4.4.1 Effect of cholesterol enriched diet on aortic diameter
A significant increase of aortic diameter (P<0.05) was found in
rabbits fed with cholesterol enriched diet as compared to that of the normal
control group. our finding is consistent with Mohammad (2008)(77) who had
found significant increase in aortic diameter (P<0.05) in rabbits fed with
cholesterol enriched diet as compared to that of the normal control group.
The increase in the aortic diameter in rabbits fed high cholesterol diet
can be explained by the expansive (enlargement) arterial remodeling which
was recognized as an important determinant in vascular pathology in which
narrowing of the lumen is the predominant feature(190). It is known that blood
vessels accommodate to the increase in blood flow by arterial growth
(Hemodynamic stimuli like flow and circumferential stress induce arterial
remodeling to achieve homeostasis of shear stress and wall tension,
respectively)(191,192). However, a decade ago it was described in macaque
monkeys that radial enlargement of vessels can also occur in response to
progressive plaque growth(193). However, Glagov found that the lumen area
of atherosclerotic human vessels remained constant until the percent stenosis
exceeded 40%. At this point lumen diameter decreased, resulting in a
restriction in flow. On the other hand he have surprising finding that
atherosclerotic arterial lumen narrowing is not simply the result of
enlargement of atherosclerotic lesions. He found instead that arteries
remodel over a large range of changes in wall mass, increasing the external
diameter in a manner that allows preservation of the arterial flow (glagoven)(194).
4.4.2 Effect of Sildenafil and Vardenafil treatment on aortic diameter
In sildenafil and vardenafil treated rabbits, there was no significant
change (P<0.05) in the aortic diameter in comparison to that of the induced
untreated group(P>0.05). Data of (Pache et al., 2002) in a study without a
control group also showed a 5.8% increase in venous and arterial diameter
after administration of 50 mg of sildenafil. The peak of the changes,
however, was observed at 30 min and by 120 min. Both arterial and venous
diameters had returned to baseline values. Polak et al. (2003) did not
observe any statistically significant effect on the retinal arterial diameters,
and no effect of sildenafil was found on a flicker-induced vasodilatation in
retinal arteries or veins. Furthermore, previous study in normal male
volunteers Grunwald et al. (2002) did not reveal any statistically significant
effect in retinal venous and arterial diameters after administration of
sildenafil(195).
This result may be due to that the increase in the aortic diameter in
rabbits fed high cholesterol diet (which can be explained by the expansive
arterial remodeling) will override the changes expected from sildenafil and
vardenafil treatment. However hemodynamic studies suggest that sildenafil
is a modest vasodilator. So it does not largely affect aortic diameter .
4.5 Effect on peak systolic blood flow velocity
4.5.1 Effect of cholesterol enriched diet on peak systolic blood flow
velocity
There was non-significant increase in aortic peak systolic velocity
(P>0.05) but significant increase in renal artery and intra-renal arteries
peak systolic velocity (P<0.05) in rabbits fed with cholesterol enriched diet
as compared to that of the normal control group. This result is in
agreement with that reported by Atsushi et al. (2003)(196) who found that
the peak systolic velocity of atherosclerotic iliac arteries of rabbits increased
significantly (2.1 times) in comparison to normal iliac arteries.
The increase in renal artery and intra-renal arteries peak systolic
velocity is due to the increased wall thickness and the development of
plaques in the arterial wall by cholesterol enriched diet feeding. When
plaques protrude in to the blood vessels, narrowing of vascular lumen
occurs which leads to very high blood flow velocity through the stenosed
region(147,148). Moreover, the increase in blood flow PSV may be due to the
fact that the atherosclerotic vessels are hardened and exhibit stiff and non
compliant state(197).
The finding that the aortic PSV was not increased significantly can be
explained by the fact that the aortic lumen is larger and it is difficult for
plaques to cause stenosis in such artery with a wide lumen to affect blood
flow velocity.
Another observation is that the aortic PSV in some rabbits was
found to decrease. This may be due to a possible development of heart
failure which perhaps caused reduction of blood pumping capability of the
heart and consequently reduction of aortic peak systolic velocity(146).
4.5.2 Effect of Sildenafil and Vardenafil treatment on peak systolic
blood flow velocity
In sildenafil and vardenafil treated rabbits there was a significant
decrease (P<0.05) in the renal artery and intra-renal artery PSV in comparison
to that of the induced untreated group, On the contrary, there was no
significant change in the aortic PSV (P>0.05) found in sildenafil treated and
vardenafil treated rabbits in comparison to the induced untreated group. These
findings are in agreement with those reported by Ercan et al. (2005) who
concluded that Sildenafil citrate had no significant effect on aortic and SMA
circulation and only caused mild changes in the carotid artery circulation.
These alterations may be considered clinically insignificant (198). Additionally
this result is consistent with that observed by Ardicoglu et al. (2005) who
demonstrated that peroral sildenafil citrate usage had slight effects on
hemodynamic parameters of the lower segmental branch of right renal
artery(199).
The reduction of renal artery and intra-renal artery peak systolic
velocities may be partly due to the fact that the treatment with The PDE5
inhibitor sildenafil and vardenafil has a double vascular muscle relaxant
potential, that is an indirect mechanism dependent on NO-cGMP and a direct
potential possibly via the inhibition of Ca2_ influx through receptor operated
and voltage dependent Ca2_ channels(200). It is possible that the vasodilator
action of sildenafil could potentially release endogenous mediators of
reconditioning from endothelial cells, such as adenosine or bradykinin, which
may trigger a signaling cascade through kinase action, resulting in NO
synthase phosphorylation and NO release(200) .
This could mean that sildenafil produces a positive effect by acting
directly on the renal dysfunctional endothelium and improving its function but
without collateral systemic effects (200).
4.6 Effect on End diastolic blood flow velocity
4.6.1 Effect of cholesterol enriched diet on end diastolic blood flow
velocity
There was unsignificant (P>0.05) decrease in aortic end diastolic
velocity and non significant (P<0.05) increase in renal artery and intrarenal arteries end diastolic velocity in rabbits fed with cholesterol enriched
diet as compared to that of the normal control group. This finding is
consistent with Mohammad (2008)(77) who found that cholesterol enriched
diet results in significant increase (P<0.05) in aortic diameter , intima-media
thickness, PI, RI, renal artery PSV, PI, RI, intra-renal artery PSV, PI, and RI
in comparison to the normal control group. There was no significant change
(P>0.05) in aortic PSV and EDV, and no significant change (P>0.05) in
renal artery and intra-renal artery EDV.
These results may be explained as that the end diastolic velocity is
not necessarily increased in stenosed arteries (normal or even low end
diastolic velocity does not exclude critical vascular stenosis). EDV may be
increased only in high degree of stenosis(201).
4.6.2 Effect of Sildenafil and Vardenafil treatment on end diastolic
blood flow velocity
Treatment of rabbits with sildenafil and vardenafil did not result in a
significant change (P>0.05) in aortic EDV, but there was a significant
decrease (P<0.05) in the renal artery and intra-renal artery EDV in
comparison to that of the induced untreated group. This finding is consistent
with that reported by Ercan et al. (2005)(198) who concluded that Sildenafil
citrate had no significant effect on aortic and SMA circulation and only
caused mild changes in the carotid artery circulation. This finding also
explained by Ardicoglu et al. (2005)(199) who demonstrated that peroral
sildenafil citrate usage had slight effects on hemodynamic parameters of
lower segmental branch of right renal artery(199).
4.7 Effect on resistive index (RI)
4.7.1 Effect of cholesterol enriched diet on resistive index
There was a significant increase (P<0.05) in aortic, renal artery and
intra-renal arteries blood flow resistive index (RI) in rabbits fed with
cholesterol enriched diet as compared to that of the normal control. This
finding is consistent with Mohammad (2008)(77) who found that cholesterol
enriched diet results in significant increase (P<0.05) in aortic diameter ,
intima-media thickness, PI, RI, renal artery PSV, PI, RI, intra-renal artery
PSV, PI, and RI in comparison to the normal control group.
This result can be explained by the fact that resistive index (RI) is a
measure of resistance in the circulation and an increase in resistive index
associated with arterial stiffness (which reflects atherosclerosis). Therefore,
increased RI is associated with the severity of systemic atherosclerosis (137).
4.7.2 Effect of Sildenafil and Vardenafil treatment on resistive index
In sildenafil treated and vardenafil treated rabbits there was
non significant decrease (p>0.05) in aortic blood flow RI but a significant
decrease (P<0.05) in renal artery and intra-renal arteries blood flow RI in
comparison to that in the induced untreated group. This finding is consistent
with (Lepore et al.) in patients with pulmonary hypertension are of
particular interest . Sildenafil produced acute reductions in pulmonary artery
pressure and pulmonary vascular resistance(87). These result are in agreement
with Ercan et al. (2005)(198). Furthermore this finding in agreement with
Enrique (2009)(200) who found that sildenafil increase significantly renal
vascular flow and significantly decrease renal vascular resistance.
The increased RI is associated with arterial stiffness and severity of
atherosclerosis. So the treatment of rabbits with sildenafil or vardenafil
results in reduction of RI. This is due to the fact that treatment with PDEIs
results in reduction in the severity of atherosclerosis .
This action on wave reflection properties implies that sildenafil
reduces arterial pulse wave velocity and causes the reflected wave to be
delayed in returning to the heart. This delay in arrival of the reflected wave
reduces its amplitude, decreases systolic and pulse pressure, and reduces LV
after load and myocardial oxygen demand. Because vasoactive drugs have
little direct effect on large elastic arteries, the wave reflection action of
sildenafil is likely confined to the muscular arteries.(202 ).
4.8 Effect on pulsatality index (PI)
4.8.1 Effect of cholesterol enriched diet on pulsatality index
There was a significant increases (P<0.05) in aortic, renal artery and
intra-renal arteries blood flow pulsatality indices in rabbits fed with
cholesterol enriched diet as compared to that in the normal control. This
finding is consistent with Mohammad (2008)(77) who found that cholesterol
enriched diet results in significant increase (P<0.05) in aortic diameter ,
intima-media thickness, PI, RI, renal artery PSV, PI, RI, intra-renal artery
PSV, PI, and RI in comparison to the normal control group.
This result can be explained by the fact that pulsatality indices (PI) is
a parameter closely related to the resistive index (RI). It has been shown to
be related to vascular resistance. An increase in pulsatality index is
associated with arterial stiffness and atherosclerosis(203) .
4.8.2 Effect of Sildenafil and Vardenafil treatment on pulsatality index
In sildenafil treated and vardenafil treated rabbits, there was non
significant decrease (p>0.05) in aortic blood flow pulsatality index but
significant decrease (P<0.05) in renal artery and intra-renal arteries blood
flow pulsatality indices in comparison to that in the induced untreated
group. This finding is consistent with (Lepore et al.) (87).As well these result
in agreement with Ercan et al. (2005)(198).
The increased PI is associated with arterial stiffness and severity of
atherosclerosis, therefore treatment of rabbits with sildenafil or vardenafil
results in reduction in PI. This is due to the fact that treatment with PDEIs
results in reduction in the severity of atherosclerosis . This action on wave
reflection properties implies that sildenafil reduces arterial pulse wave
velocity and causes the reflected wave to be delayed in returning to the
heart. This delay in arrival of the reflected wave reduces its amplitude,
decreases systolic and pulse pressure, and reduces LV after load and
myocardial oxygen demand. Because vasoactive drugs have little direct
effect on large elastic arteries, the wave reflection action of sildenafil is
likely confined to the muscular arteries.(202 ).
4.9 Effect on renal artery-to-aortic peak systolic
velocity ratio (RAR)
4.9.1 Effect of cholesterol enriched diet on renal artery–to-aortic peak
systolic velocity ratio (RAR)
There was significant increase (P<0.05) in renal artery –to-aortic
peak systolic velocity ratio (RAR) in rabbits fed with cholesterol enriched
diet as compared to the control of the same group.
The explanation of this runs as follows: because there was a
significant increase in the PSV of renal artery and non significant rise in
the PSV of aorta, a significant increase in the RAR takes place which is an
important Doppler index used in the diagnosis of renal artery stenosis (130).
4.9.2 Effect of Sildenafil and Vardenafil treatment on renal artery –toaortic peak systolic velocity ratio (RAR)
In sildenafil treated and vardenafil treated rabbits, there was non significant
decrease (p>0.05) in renal artery –to- aortic peak systolic velocity ratio in
comparison to that in the induced untreated group.
The reduction of RAR observed by vardenafil and sildenafil
treatment was because the treatment with both drugs leads to a significant
reduction in the PSV of renal artery but no significant reduction in the PSV
of aorta. The reduction in RAR was not significant perhaps due to high
cholesterol diet continued with the treatment which prevents the optimal
action of the drugs or perhaps due to small sample size studied. Another
reason for the non significant reduction in RAR may be due to the low doses
of drugs and the short duration of treatment.
4.10 Effect on aortic histological change
4.10.1 Effect of cholesterol enriched diet on aortic histology
All rabbits (100%) fed with cholesterol enriched diet had an
involvement of their aorta with different phases of atherosclerotic lesions at
the end of 12 weeks of the study that was significantly different (P<0.05) as
compared with the normal control group. This finding is in agreement with
that reported by Geetha et al. (2007)(204) and Asgary et al. (2007)(205) these
finding might be due to atherogenic diet which encouraged atherosclerotic
changes on aortic wall.
4.10.2 Effect of
histology
Sildenafil and Vardenafil treatment on aortic
The overall histopathological features of rabbits treated with
sildenafil and verdenafil are better than that of induced untreated group but
this differences in severity of atherosclerotic changes dose not reach
significant level (P>0.05).
This finding may be due to the fact that rabbits were continued to be
fed with cholesterol enriched diet together with the treatment with
sildenafil and verdenafil which may prevent the maximal effect of the
drugs or due to low doses of drugs and short duration of treatment. although
sildenafil and vardenafil have the ability to improve endothelial function (206),
and possess antioxidant action(186), additionally sildenafil might have an
antiangiogenic effect, since sildenafil, tadalafil, and vardenafil have a
beneficial effect on inflammatory activation and surrogate markers of
endothelial dysfunction(206).
5.1 Conclusions
The following points can be concluded out of this study:
1. High cholesterol diet causes a significant increase in aortic diameter,
intima-media thickness, RI and PI and a significant increase in renal
artery and intra-renal arteries blood flow PSV, PI and RI in male
rabbits.
2. Sildenafil and Vardenafil did not significantly change the serum level of
TC, TG, LDL-C, VLDL-C, HDL-C and the level atherogenic index in
treated animals.
3. Both drugs significantly reduce the serum level of MDA and increase the
serum level of GSH.
4. Both drugs did not significantly change the aortic intima-media
thickness and aortic diameter of treated animals.
5. Both drugs significantly increase the arterial elasticity and decrease
arterial stiffness as indicated by reduction of RI and PI of the aorta,
and PSV,EDV,RI,PI of renal artery and intra-renal arteries.
5.2 Recommendations
1. In view of the effect of vardenafil and sildenafil in reducing of arterial
resistance and stiffness as indicated by their ability to decrease PSV, PI
and RI and have anti-oxidant effect. So further experimental studies with
measurement of effect of vardenafil and sildenafil on inflammatory
markers and cytokines which have major role in pathogenesis of
atherosclerosis.
2. Because vardenafil and sildenafil
treatment results in significant
reduction in renal artery peak systolic velocity and non significant
reduction in renal artery –to-aortic peak systolic velocity ratio which are
Doppler indices used in the diagnosis of renal artery stenosis. So further
experimental studies with large sample size and long periods about the
effectiveness of vardenafil and sildenafil in treatment of renal artery
stenosis may be required .
3. Different doses of vardenafil and sildenafil and combination with other
PDEIs essential to reveal their effects on progression of atherosclerosis.
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‫الخالصة‪:‬‬
‫يعتبر مرض تصلب الشرايين القاتل الرئيسي على مستوى العالم‪ .‬ويعتبر ارتفاع مستوى‬
‫الدهون بالدم‪ ,‬مرض السكري‪ ,‬ارتفاع ضغط الدم الشرياني‪ ,‬التدخين والسمنة من أهم‬
‫عوامل الخطورة للمرض‪ .‬أن)‪ )sildenafil and vardenafil‬هي من األدوية‬
‫األساسية التي تستخدم في عالج العجز الجنسي و من األدوية الواعده التي تستخدم في‬
‫عالج تصلب الشرايين ‪.‬‬
‫الطريقة‪:‬‬
‫اربعه وعشرون أرنبا محليا من الذكور أدخلت في هذه الدراسة‪ .‬تم توزيع هذه األرانب‬
‫بصورة عشوائية إلى أربعة مجاميع ‪ :‬المجموعة األولى هي مجموعة السيطرة الطبيعية‬
‫وأعطيت غذاء قياسي طبيعي لمدة اثنا عشر أسبوعا‪ .‬المجموعة الثانية أعطيت غذاء‬
‫عالي الدسم يحتوي ‪ %2‬كولسترول لمدة اثنا عشر أسبوعا‪ .‬المجموعة الثالثة أعطيت‬
‫غذاء عالي الدسم لمدة ستة أسابيع‪ ,‬بعد ذلك أعطيت عقار السلدنافيل ‪5‬ملم‪/‬كغم لكل يوم‬
‫عن طريق الفم بالموازاة مع الغذاء العالي الدسم لمدة ستة أسابيع أخرى‪ .‬المجموعة‬
‫الرابعة أعطيت غذاء عالي الدسم لمدة ستة أسابيع‪ ,‬بعد ذلك أعطيت عقار الفيردنافيل‬
‫‪ 18‬ملم‪/‬كغم لكل يوم عن طريق الفم بالموازاة مع الغذاء العالي الدسم لمدة ستة أسابيع‬
‫أخرى‪ .‬تم سحب عينات الدم أوال عند بداية الدراسة‪ ,‬وعند ستة أسابيع من فترة الدراسة‬
‫ثم كل أسبوع خالل فترة المعالجة البالغة ستة أسابيع‪ .‬تم قياس مستوى الدهون بالدم وهي‬
‫الكولسترول الكلي‪ ,‬الكلسيريدات الثالثية‪ ,‬الكولسترول واطئ الكثافة‪ ,‬الكولسترول واطئ‬
‫الكثافة جدا الكولسترول عالي الكثافة ‪,‬مقياس درجة التصلب‪ ,‬ومستوى أالجهاد‬
‫سمك جدار‬
‫التاكسدي في الدم المتمثل بمستوى(‪ )MDA and GSH‬تم قياس عرض و ُ‬
‫الشريان األبهر البطني وتم قياس سرعة جريان الدم (السرعة االنقباضية العليا‪ ,‬السرعة‬
‫االنبساطية النهائية‪ ,‬مقياس درجة المقاومة‪ ,‬مقياس درجة االنقباضية واالنبساطية ونسبة‬
‫السرعة االنقباضية العليا في الشريان الكلوي ‪/‬السرعة االنقباضية العليا في الشريان‬
‫األبهر) في الشريان األبهر‪ ,‬الشريان الكلوي والشرايين الداخل كلوية بواسطة جهاز‬
‫الدوبل رالمتوفر في العيادة الخاصة للدكتور عقيل زوين عند بداية الدراسة وبعد ستة‬
‫اسابيع وعند نهايتها‪ .‬كذلك تم فحص المقاطع النسيجية للشريان االبهر البطني عند نهاية‬
‫الدراسة ‪.‬‬
‫وكانت النتائج كاألتي‪:‬‬
‫سبب الغذاء العالي الدسم الذي يحتوي ‪ %2‬كولسترول زيادة معنوية (‪ )P<0.05‬في‬
‫مستوى الكولسترول الكلي‪ ,‬الكلسيريدات الثالثية‪ ,‬الكولسترول واطئ الكثافة‪,‬‬
‫الكولسترول واطئ الكثافة جدا‪ ,‬الكولسترول عالي الكثافة‪ ,‬وسبب زيادة معنوية‬
‫(‪ )P<0.05‬في مقياس درجة التصلب‪ .‬كما سبب زيادة معنوية (‪ )P<0.05‬في مستوى‬
‫النخفاض‬
‫أالجهاد التاكسدي في الدم المتمثل )بارتفاع مستوى ‪ MDA‬المصاحب‬
‫سمك الشريان االبهر و‬
‫مستوى‪ .) GSH‬كان هناك أيضا زيادة معنوية في عرض و ُ‬
‫مقياس درجة مقاومتها ومقياس درجة االنقباضية واالنبساطية فيها كما سبب زيادة‬
‫معنوية (‪ )P<0.05‬في السرعة االنقباضية العليا‪ ,‬مقياس درجة المقاومة ومقياس درجة‬
‫االنقباضية واالنبساطية في الشريان الكلوي والشرايين الداخل كلوية بالمقارنة مع‬
‫مجموعة السيطرة الطبيعية‪ .‬كما سبب زيادة معنوية (‪ )P<0.05‬في )نسبة السرعة‬
‫االنقباضية العليا في الشريان الكلوي ‪/‬السرعة االنقباضية العليا في الشريان األبهر( ‪.‬لم‬
‫يسبب الغذاء العالي الدسم زيادة معنوية (‪ )P>0.05‬في السرعة االنقباضية العليا‬
‫والسرعة االنبساطية النهائية في الشريان األبهر كما لم يسبب زيادة معنوية (‪)P>0.05‬‬
‫في السرعة االنبساطية النهائية في الشريان الكلوي والشرايين الداخل كلوية‪.‬‬
‫بالنسبة لنتائج الفحص النسيجي فان جميع األرانب التي تغذت على الغذاء العالي الدسم‬
‫ظهر فيها مراحل مختلفة من تصلب الشرايين وكان هناك زيادة معنوية في نسبة‬
‫التصلب (‪ )P<0.05‬بالمقارنة مع مجموعة السيطرة الطبيعية ‪.‬‬
‫لم يسبب العالج بالعقاران )‪ )sildenafil and vardenafil‬انخفاضا معنويا‬
‫(‪ ) P<0.05‬في الكولسترول الكلي‪ ,‬الكلسيريدات الثالثية ‪ ,‬الكولسترول واطئ الكثافة‪,‬‬
‫الكولسترول العالي الكثافة‪ ,‬الكولسترول واطئ الكثافة جدا في الدم ومقياس درجة‬
‫التصلب‪ .‬بينما سبب العقاران )‪)sildenafil and vardenafil‬زيادة معنوية‬
‫(‪ )P<0.05‬في مستوى (‪ )GSH‬و انخفاضا معنويا في مستوى (‪ )MDA‬في الدم‪.‬‬
‫كما لم يسبب العالج بالعقاران )‪)sildenafil and vardenafil‬انخفاضا معنويا‬
‫سمك جدار الشريان االبهر البطني‪.‬ولم يسبب العالج بالعقاران‬
‫(‪ )P<0.05‬في عرض و ُ‬
‫)‪)sildenafil and vardenafil‬انخفاضا معنويا (‪ )P>0.05‬في السرعة االنقباضية‬
‫العليا‪ ,‬السرعة االنبساطية النهائية‪ ,‬مقياس درجة المقاومة ومقياس درجة االنقباضية‬
‫واالنبساطية في الشريان األبهر‪ ,‬ولم يسبب العالج بالعقاران ‪sildenafil and‬‬
‫)‪)vardenafil‬انخفاضا معنويا (‪ )P>0.05‬في) نسبة السرعة االنقباضية العليا في‬
‫الشريان الكلوي ‪/‬السرعة االنقباضية العليا في الشريان األبهر( ‪,‬بينما سبب العالج‬
‫بالعقاران )‪ )sildenafil and vardenafil‬انخفاضا معنويا (‪ )P<0.05‬في السرعة‬
‫االنقباضية العليا‪ ,‬السرعة االنبساطية النهائية‪ ,‬مقياس درجة المقاومة ومقياس درجة‬
‫االنقباضية واالنبساطية في الشريان الكلوي والشرايين الداخل كلوية بالمقارنة مع‬
‫مجموعة السيطرة الغير معالجة‪.‬‬
‫بالنسبة لنتائج الفحص النسيجي العقاران )‪)sildenafil and vardenafil‬سببا انخفاضا‬
‫في نسبة التصلب الشرياني ولكن هذا االنخفاض لم يكن انخفاضا معنويا (‪)P>0.05‬‬
‫بالمقارنة مع مجموعة السيطرة الغير معالجة‪.‬‬
‫لم يكن هناك اختالف معنوي (‪ )P>0.05‬في الباراميترات المقاسة بين مجموعة‬
‫األرانب التي عولجت بعقار ‪ sildenafil‬ومجموعة األرانب التي عولجت بعقار‬
‫‪. vardenafil‬‬
‫االستنتاج ‪:‬‬
‫لم يسبب العقاران )‪ )sildenafil and vardenafil‬انخفاضا في مستوى الكولسترول‬
‫الكلي‪ ,‬الكلسيريدات الثالثية ‪ ,‬الكولسترول واطئ الكثافة‪ ,‬الكولسترول واطئ الكثافة‬
‫جدا ‪ ,‬الكولسترول العالي الكثافة في الدم‪ ,‬كما لم يسببان انخفاضا في مقياس درجة‬
‫سمك جدار الشرايين في األرانب المعالجة بهذين‬
‫التصلب‪ ,‬شدة تصلب الشرايين و ُ‬
‫العقاريين ‪,‬بينما سبب العقاران )‪)sildenafil and vardenafil‬زيادة معنوية‬
‫(‪ )P<0.05‬في مستوى (‪ )GSH‬و انخفاضا معنويا في مستوى (‪ )MDA‬في الدم‪ .‬لهذا‬
‫فان هذين العقارين غيرا من قوة وصالبة الشرايين كما تبين من قدرتهما على تخفيض‬
‫مقياس درجة المقاومة ومقياس درجة االنقباضية واالنبساطية في الشريان االبهر‬
‫البطني‪,‬وكدلك تبين امكانية العقارين في تخفيض مقياس السرعة االنقباضية العليا‪,‬‬
‫السرعة االنبساطية النهائية‪ ,‬مقياس درجة المقاومة ومقياس درجة االنقباضية‬
‫واالنبساطية في الشريان الكلوي والشرايين الداخل كلوية في األرانب المعالجة‪.‬‬
‫دراسة‬
‫س ُم ْك الطبقة الوسطية و الداخلية‬
‫ثأثير السلدينافيل والفيردينافيل على ُ‬
‫للشريان األبهر وعلى سرعة جريان الدم في الشريان األبهر‬
‫‪,‬الشريان الكلوي و الشريان الكلوي الداخلي في ذكور األرانب‬
‫ذات مستوى الدهون المرتفع‬
‫رسالة‬
‫مقدمة إلى مجلس كلية الطب‪ /‬جامعة الكوفة‬
‫كجزء من متطلبات‬
‫نيل درجة الماجستير في األدوية و العالجيات‬
‫من قبل‬
‫الصيدالني كرار حسين حريب كمونه‬
‫بكالوريوس علوم صيدلة‬
‫المشرف‬
‫المشرف‬
‫د‪ .‬نجاح رايش الموسوي‬
‫د‪.‬عقيل زوين‬
‫أستاذ في علم األدوية والعالجيات‬
‫مدرس في علم فسلجة‬
‫القلب و األوعية الدموية‬
‫‪1430‬هــ‬
‫‪ 2009‬م‬
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