Effect of Hemodynamic Forces on
Arterial Wall Transport, Biochemical
and Structural Properties
by
Syed Yawer Husain
Centre for Biomedical Engineering
Submitted
In fulfillment of the requirements of the degree of
DOCTOR OF PHILOSOPHY
to the
Indian Institute of Technology, Delhi
October 2003
fli
676
CERTIEICA`IE
This is to certify that the thesis entitled "Effect of Hemodynamic Forces on
Arterial Wall Transport, Biochemical and Structural properties" being
submitted by Syed Yawer Husain for the award of the degree of DOCTOR OF
PHILOSOPHY, is a record of the original bonafide research work carried out by
him. He has worked under our joint guidance and supervision and has fulfilled
the requirement for the submission of this thesis. The results presented in this
thesis have not been submitted in part or full to any other university or institute
for award of any degree or diploma.
D . Alok Ray
Professor
Centre for Biomedical Engineering
ha ,r Ali
Reader
Department of Biochemistry
Indian Institute of Technology
Hamdard University
New Delhi-1100 16.
New Delhi-1100 62
ACKNOWLEDGEMENTS
It requires a Shakespeare's pen to write exactly what one feels. I
compulsively feel in this instance, to gratefully acknowledge the people
who motivated, guided and assisted me in one way or other.
I feel very proud to have the privilege to work under the guidance
of Prof. Alok Ray, who had shown the light to me all the time. His
fatherly wisdom and constant encouragement, without which this work
would have been next to impossible, was a source of inspiration. I also
appreciate the confidence he had imbued in me to work during my
course of Ph.D. It is to him that I owe a great share of what I am today.
I also feel pleasure in expressing my deep sense of gratitude to my
joint supervisor, Dr. Shakir Ali, who has been my teacher also. I am
grateful to him for teaching me punctuality, discipline, sense of
responsibility and hard work. His constant indulgence was a source of
encouragement in pursuing my research work. I thank him for rendering
insightful suggestions, probing comments, generous support and
guidance time to time.
The center with all its faculty members has been able to provide
the necessary services for the work. I express my sincere gratitude to
Prof. Harpal Singh, Head, Center for Biomedical Engineering for his
sincere advice and constant encouragement.
I would like to express my sincere thanks to Dr. Prashant Mishra,
DBEB, IIT Delhi for his valuable suggestions.
I extremely obliged to Dr. A.K. Dinda, Department of Pathology,
AIIMS, New Delhi for permitting me to carry out confocal laser
microscopy and for his critical analysis of the pathological approach of
my study. Thanks also reserved for Dr. T.K. Das, Department of
Anatomy, AIIMS, New Delhi for providing me necessary support and for
his expert opinion.
I am also thankful to all my Lab colleagues at IIT Delhi as well as
Hamdard University, for their love and affection. They made my stay in
the lab a pleasant one and have never been away for helping me at all
times.
Friends are the most important part of life who are there in good as
well as bad times therefore. I am lucky to have been in the company of
good and faithful friends, whom I shared all my smile and fears.
Much is owed to my father beyond words can express. I am highly
thankful to Almighty, who has gifted me with caring, loving and
supportive father who stood besides me always and strengthened my
shaking confidence. I also express my heartfelt gratitude to all my family
members for their unfaltering support, care, immense love and affection
that was a constant source of encouragement to me.
I also wish to acknowledge all those people whose name could not
be mentioned but have helped me in this journey.
The financial assistance from CSIR in the form JRF and SRF is
gratefully acknowledged.
[Syed Yawer Husain]
irkinealthr
An eternal shower of love...
Abstract
Arterial wall respond to hemodynamic forces, such as fluid shear stress and
blood pressure, generated by blood and its viscous flow. In the present study, the
relative contribution of hydrostatic pressure (70 and 150 cm water pressure,
which represents normo- and hypertensive physiological situations, respectively)
and fluid shear stress (pulsatile/laminar) on macromolecular transport across the
rabbit aortic wall, its redox state and on its structural properties is determined. An
experimental in vitro model that allows adjustment of the hydrostatic pressure
with pulsatile or laminar flow in the rabbit thoracic aorta was designed/fabricated
and used. The entire study was done on cannulated aortic segments.
Hemodynamic forces largely affect transport properties of arterial wall with
respect to macromolecular transport. Pulsatile shear stress with higher pressure,
cause alterations in vessel wall transport. These alterations in macromolecular
trafficking across vessel wall may help understand the development of vascular
pathology like atherosclerosis. The study involving hemodynamic forces-induced
oxidative stress in vessel wall provides evidence for an atheroprotective role of
laminar shear stress in normal physiological conditions. Pulsatile shear stress in
combination with hydrostatic pressure is observe to be a weak inducer of
antioxidant defense in blood vessels. These findings could be useful in future to
consider the issue of hypertension-mediated free radical generation in the vessel
wall and possible therapeutic role of antioxidants. Pulsatile shear stress, in
combination with pressure, has been found to produce significant structural
alteration in aortic wall. Increased surface expression of platelet/endothelial
adhesion molecule, PECAM-1 with respect to pulsatile shear stress and pressure
may explain mechanoresponsive role of PECAM-1 in vessel wall.
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TABLE OF CONTENTS
Page No.
Certificate
Acknowledgements
Abstract
List of figures
vi
List of tables
1. Introduction
1-9
1.1. Role of hemodynamic forces in arterial wall pathology
1.2. Biochemical basis of hemodynamic forces-induced arterial wall response
1.3. Present status and lacunae in the study
1.4. Scope of the Thesis
2. Review of literature
10-61
2.1 Hemodynamics and arterial wall
2.1.1. Components of arterial wall hemodynamics
2.1.1.1. Wall shear stress
2.1.1.2. Hydrostatic pressure or circumferential stretch
2.2 Hemodynamics and arterial wall pathology
2.3 Functional response of arterial wall to hemodynamic forces
2.3.1 Transport of macromolecules across the vessel wall
2.3.1.1 Vasculopathy associated with transmural
transport of macromolecules
2.3.2. Biochemical response of arterial wall
2.3.2.1. Endothelial Nitric Oxide
2.3.2.2. Arterial wall oxidative stress
2.4. Hemodynamic forces induced arterial wall structural alterations
2.4.1 Phenotypic Change in the vessel wall
2.4.1.1 Hemodynamic oxidative stress and wall growth
2.4.1.2 Expression of cell adhesion molecules
ii
62-83
3. Materials and Methods
3.1. Solutions and chemicals
3.2. Animal Model
3.3. Instruments
3.4. Perfusion system
3.5. Artery preparation
3.6. Experimental hemodynamic conditions
3.6.1. To study the effect of laminar shear stress and pressure
3.6.2. To study the effect of pulsatile shear stress and pressure
3.7. Estimation of tracer uptake and flux
3.8. Biochemical estimations
3.8.1. Processing of the tissue and preparation of lysate (homogenate)
3.8.2. Sub-cellular Fractionation
3.8.3. Enzyme estimations
3.8.3.1. Xanthine Oxidase (XO)
3.8.3.2. Lipid Peroxidation (LPO)
3.8.3.3. Reduced glutathione (GSH)
3.8.3.4. Superoxide Dismutase (SOD)
3.8.3.5. Glutathione reductase (GR)
3.8.3.6. Glutathione peroxidase (GPx)
3.8.3.7. Catalase (CAT)
3.8.3.8. Glutathione S-tranferase (GST)
3.8.3.9. Glucose 6-phosphate dehydrogenase (G6PD)
3.8.4. Estimation of protein
3.9. Electron Microscopy
3.9.1. Tissue preparation for transmission and scanning electron microscopy
3.9.1.1. Scanning Electron microscopy
3.9.1.2.Transmission electron microscopy
3.10. Light Microscopy
3.10.1. Fixation and Processing
3.10.2. Section Cutting
3.10.3. Staining (Hematoxylin and Eosin)
3.10.3.1. Reagents
3.11. Confocal Laser Microscopy
3.11.1. Procedure
3.11.1.1. Quantification of PECAM-1 expression
iii
4. Effect of Pulsatile/Laminar Shear Stress and Pressure
on Aortic Wall Macromolecular Transport
84-97
4.1. Introduction
4.2. Experimental design
4.2.1. Hemodynamic flow conditions
4.3. Results
4.3.1. Statistical analysis
4.4. Discussion
5. Endothelial Nitric oxide production under Laminar/ pulsatile
shear stress and pressure
98-110
5.1. Introduction
5.2. Experimental design
5.2.1. Studies involving laminar shear stress and pressure
5.2.2. Studies involving pulsatile shear stress and pressure
5.3. Estimation of nitric oxide
5.4. Results
5.5. Discussion
6. Hemodynamic forces-induced biochemical changes in
aortic wall: effect on redox state of the tissue
6.1. Introduction
6.2. Experimental conditions
6.3. Results
6.3.1. Effect of hemodynamic forces on wall oxidative stress
6.3.1.1. Superoxide dismutase (SOD) activity
6.3.1.2. Catalase activity
6.3.1.3. Glutathione peroxidase (GPx)
6.3.1.4. Glucose-6-phosphate dehydrogenase (G6PD)
6.3.1.5. Glutathione reductase (GR)
6.3.1.6. Glutathione-s-transferase (GST)
6.3.1.7. Reduced glutathione (GSH)
6.3.1.8. Lipid peroxidation (LPO)
6.3.1.9. Xanthine oxidase (XO)
6.3.2. Statistical Analysis
6.4. Discussion
iv
111-132
7. Effect of hemodynamic stresses on aortic wall structure
133-146
7.1. Introduction
7.2. Experimental conditions
7.3. Results
7.3.1. Light microscopy of aortic wall
7.3.2. SEM of normal endothelial surface
7.3.3. SEM on aorta under laminar shear stress and pressure
7.3.4. SEM analysis of aorta under pulsatile shear stress and pressure
7.3.5. TEM observation on endothelial layer and sub-endothelial layer
7.3.5.1. TEM on aorta exposed to laminar shear stress and pressure
7.3.5.2. TEM on aorta under pulsatile shear stress and pressure
7.4. Discussion
8. Summary and conclusions
147-153
Bibliography
154-188
Brief biodata
189
List of publication
v