AU-KBC Life Sciences - Vascular Biology Lab

Haemodynamic forces are important modulators of vascular tone and vascular wall remodelling, and are increasingly implicated in many physiological and pathological functions. Blood vessels are under the influence of two primary haemodynamic forces: The circumferential force - the wall tension by the blood pressure and, secondly, the frictional force or shear stress which results from blood flow along the vessel wall. The shear stress experienced by the endothelium is a function of the ‘axial’ pressure, which occurs as blood flows through the vessel and, physiologically, is of the order of 0–50 dyn/cm2. Shear stress could be transduced into a wide range of associated intracellular biochemical events. Blood flow was first recognized to be important in the control of vascular tone in 1933 (Schretzenmayr, 1933), in femoral artery of dog. It is now recognized that the endothelial cell, uniquely situated at the interface between the blood and the vascular wall, is effectively a biological mechanotransducer which senses shear forces and converts these physical stimuli to intracellular biochemical signals.

Shear stress appears to be a particularly important hemodynamic force because it stimulates the release of vasoactive substances and changes gene expression, cell metabolism, and cell morphology. The nature and magnitude of shear stress plays an important role in long-term maintenance of the structure and function of the blood vessel. While steady shear stress generally stimulates many of the same endothelial cell responses as pulsatile stress, oscillatory shear stress leads to pathological conditions like atherosclerosis, through endothelial damage. Areas of low shear stress may be more prone to intimal lesion formation, because in these areas the endothelium may produce less vasoprotective factors, such as NO and prostacyclin unlike normal shear stress. Considering the beneficial effects of NO and prostacyclin, it could be speculated that the spatial distribution of atherosclerotic plaques in areas of low shear stress may in part be explained by a reduced elaboration of NO (decrease in eNOS) and prostacyclin at these sites. This reduction in NO levels leads to decrease in the endothelial functions.

Cardiovascular diseases, particularly ischemic heart disease and myocardial infarction, are the major concerns presenting a major scientific challenge for the development of optimal therapies.The mechanosensors on the endothelium that sense changes in blood flow and shear stress are poorly defined at the molecular level. Our lab focuses on shear stress mediated regulation of functional and mechanistic aspects of endothelium and blood vessels.

Physics Of Shear stress - Strain - Stress - Types of Stress

SS- Pathology - Implication - Cardiovascular blood flow - Atherosclerosis - Portal hypertension

PHYSICS OF SHEAR STRESS

STRAIN

Strain is the geometrical expression of deformation caused by the action of stress on a physical body. Strain therefore expresses itself as a change in size and/or shape. When a body is subjected to a load (force), it is distorted or deformed. If the load is small, the distortion will probably disappear when the load is removed. Such a proportional dimensional change-intensity, or degree, of distortion is known as strain.

If strain is equal over all parts of a body, it is referred to as homogeneous strain; otherwise, it is inhomogeneous strain. Strain, $\epsilon \;$ is given by

Elastic strain is a transitory dimensional change that exists only while the initiating stress is applied and disappears immediately upon removal of the stress.

Plastic strain is a dimensional change that does not disappear when the initiating stress is removed.

STRESS

Stress is the internal resistance, or counterforce, of a material to the distorting effects of an external force or load. These counterforces tend to return the atoms to their normal positions. The total resistance developed is equal to the external load. This resistance is known as stress.

Stress (s) can be equated to the load per unit area or the force (F) applied per cross-sectional area (A) perpendicular to the force. The external load and the area to which it is applied can be measured by the equation below:

TYPES OF STRESS

Stresses occur in any material that is subject to a load or any applied force. There are many types of stresses, but they can all be generally classified in one of six categories: residual stresses, structural stresses, pressure stresses, flow stresses, thermal stresses, and fatigue stresses.

Fatigue stresses are due to cyclic application of a stress. The stresses could be due to vibration or thermal cycling. When loadings are cyclic or unsteady, stresses can effect a material more severely. Stress intensity within the body of a component is expressed as one of three basic types of internal load. They are known as tensile, compressive, and shear.

Mathematically, there are only two types of internal load because tensile and compressive stress may be regarded as the positive and negative versions of the same type of normal loading. This is illustrated in the following diagram:

Tensile stress is that type of stress in which the two sections of material on either side of a stress plane tend to pull apart or elongate as illustrated in figure (A).

Compressive stress is the reverse of tensile stress. Adjacent parts of the material tend to press against each other through a typical stress plane as illustrated in figure (B).

Shear stress exists when two parts of a material tend to slide across each other in any typical plane of shear upon application of force parallel to that plane as illustrated in figure (C).

While the plane of a tensile or compressive stress lies perpendicular to the axis of operation of the force from which it originates, the plane of a shear stress lies in the plane of the force system from which it originates (depicted as below).

Two types of stress can be present simultaneously in one plane, provided that one of the stresses is shear stress. Under certain conditions, different basic stress type combinations may be simultaneously present in the material. An example would be a reactor vessel during operation. The wall has tensile stress at various locations due to the temperature and pressure of the fluid acting on the wall. Compressive stress is applied from the outside at other locations on the wall due to outside pressure, temperature, and constriction of the supports associated with the vessel. In this situation, the tensile and compressive stresses are considered principal stresses. If present, shear stress will act at a 45° angle to the principal stress.

SS- Pathology

Implication

Cells in situ are always subjected to varied mechanical stresses including gravitational force, mechanical stretch or strain, and shear stress. Especially the endothelial cells (ECs) in particular, which form the inner lining of the blood vessels are constantly exposed to hemodynamic forces in the form of shear stress due to the pulsatile nature of blood pressure and flow. Endothelial cells also respond differently to different modes of shear forces, e.g., laminar, disturbed, or oscillatory flows.

ECs lining the vessel wall recognize shear stresses convert mechanical stimuli into intracellular signals that affect cellular functions like proliferation, migration, permeability, cytoskeleton remodeling, and gene expression as well. ECs transduce signals to vascular muscular cells and others in order to modify vessel shape and structure accordingly.

How do cells, and the endothelium in particular, sense a change in shear stress, and what are the signaling pathways for the cellular response? From a simplistic standpoint, changes in fluid shear stress could be sensed directly by cell membrane components such as membrane proteins, ion channels, or caveolae or by alterations of the cellular cytoskeleton; subsequent cellular signaling cascades through phosphorylation events or generation of reactive oxygen species (ROS) can lead to diverse effects such as the release of cytokines and other mediators, activation of transcription factors, altered gene and protein expression, and cell division or death.

In vitro studies on cultured ECs in flow channels have been conducted to investigate the molecular mechanisms by which cells convert the mechanical input into biochemical events, which eventually lead to functional responses. The knowledge gained on mechano-transduction, with verifications under in vivo conditions, will advance our understanding of the physiological and pathological processes in vascular remodeling and adaptation in health and disease.

Cardiovascular blood flow

Different forms of heart disease can be caused by atherosclerosis (hardening or furring of the arteries). Atherosclerosis is caused by the formation of plaques that bulge into the artery, narrowing the blood vessel. Cardiovascular diseases, particularly ischemic heart disease and myocardial infarction, are the major concerns presenting a major scientific challenge for the development of optimal therapies.

Cerebral strokes, another vascular disorder, are another points of concern. These vascular diseases share a common factor - atherosclerosis and the failure or destruction of the vascular wall structure. This mechanical disruption of the vascular wall specifically the arterial wall, leads to fatal outcomes such as acute coronary syndrome and sub-arachnoid hemorrhage. Therefore, both the onset and final consequences of tragic fatal vascular diseases are inseparably connected to mechanical events that occur on the vascular wall, which are likely influenced by alterations in blood flow.

Atherosclerosis

Atherosclerosis is an inflammatory process disease that involves the artery wall and is characterized by the progressive accumulation of lipids, cholesterol, calcium, and cellular debris within the intima of the vessel wall, endothelial dysfunction and vascular inflammation. This buildup results in plaque formation, vascular remodeling, acute and chronic luminal obstruction, abnormalities of blood flow, and diminished oxygen supply to target organs. Atherosclerosis preferentially occurs at specific arterial sites, branches, bifurcations and bends, and this phenomenon is thought to be related to hemodynamics.

One factor that determines how easily an atherosclerotic plaque forms is the wall shear stress that the blood flow puts on the walls of the blood vessels. Under normal shear stress a normal production of Nitric oxide, prostacyclin, thrombomodulin and Plasminogen activator is evident, which acts as Anti-thrombic agents, while NO and TGF-ß are anti Atherosclerotic.

With low shear stress (LSS) and oscillatory shear stress ( OSS) being pro-atherogenic. It was shown that LSS induces the production of more IP-10, GRO-alpha (chemotactic factors) and fractalkine (membrane-bound soluble protein) than OSS, which in fact induced no fractalkine. This correlated with differences in the composition of the atherosclerotic plaques: LSS induced plaques with thinner fibrous caps and larger necrotic cores than OSS, which is associated with plaque rupture that can cause heart attacks.

Low shear stress is associated with increased wall thickening and accelerated lumen narrowing in moderately diseased human coronary arteries. There is a continuous inverse relation between shear stress and rate of lumen narrowing.

Areas of low shear stress may be more prone to intimal lesion formation, because in these areas the endothelium may produce less vasoprotective factors, such as NO and prostacyclin unlike normal shear stress. Considering the beneficial effects of NO and prostacyclin, it could be speculated that the spatial distribution of atherosclerotic plaques in areas of low shear stress may in part be explained by a reduced elaboration of NO (decrease in eNOS) and prostacyclin at these sites. This reduction in NO levels leads to decrease in the endothelial functions.

While low shear stress is associated with atherogenesis and disease progression, regions of moderate to high shear stress are relatively spared of intimal thickening as long as flow remains unidirectional and axially aligned.

Increase in endothelial shear stress (due to increase in flow) is associated with enlargement of non-atherosclerotic arteries, as seen in arteriovenous fistulas. The same phenomenon may occur in atherosclerosis. As plaque impinges on the vessel lumen, endothelial shear stress increases. This might be expected to trigger the same remodeling response that occurs in other settings of increased shear stress.

Portal hypertension

Portal hypertension is defined by an elevation in blood pressure in the portal system. Either an increase in blood flow, an increase in resistance, or both elevates portal pressure. In the normal liver, intrahepatic resistance changes with variations in portal blood flow, thereby keeping portal pressure within normal limits. In cirrhosis, however, both intrahepatic resistance and splanchnic blood flow are increased. Normally, blood is carried to the liver by a major blood vessel called the portal vein. If blood can’t flow easily through the liver because of cirrhosis, the blood gets slowed down in this vein and the pressure inside the vein increases. This higher blood pressure in the portal vein is called portal hypertension.

If blood can’t flow normally through the portal vein, it must return to the heart using other blood vessels. These vessels become swollen, called Varices because of the increased amount of blood flowing through them. Varices have thin walls and can easily break open because they aren’t meant to handle such high-pressure blood flow, which can be very fatal.

It is firmly established fact that NO production is stimulated by shear stress and shear stress and portal hypertension are related. In chronic liver injury, the molecular basis of the intrahepatic NO deficiency has uniformly been attributed to a decreased activity of the endothelial isoform (eNOS). Based on organ-specific distribution of NOS, there could be an important aspect of the role of NO in portal hypertension. Besides a decreased production, there might also be an increased degradation of NO because of enhanced superoxide activity as a result of superoxide dismutase deficiency, leading to diminished bioavailability of NO.

Modulating the hepatic system by decreased intrahepatic vascular resistance (IHVR) while maintaining or enhancing hepatic blood flow. Furthermore, the vasodilatory effect should be limited to the hepatic microcirculation to prevent a worsening in splanchnic/systemic vasodilatation.

Our Research Interest

Recent works suggest that shear stress is a biophysical stimulator for nitric oxide (NO) production, which is known to be involved in a wide range of physiological as well as pathophysiological developments including vascular remodeling. Animal, molecular, and cellular studies of the endothelium's response to hemodynamic shear stress have provided new insights into the possible link between shear stress and cellular migration. Cellular migration is highly implicated in vascular remodeling and angiogenesis. Although published data clearly show that shear stress is determinant of both NO production and migration in endothelial cells (EC) the inter-relationship between SS, NO and EC migration is not yet known.

Our aim is to unravel the mechanisms by which SS mediated migration of EC is coupled with the NO generation.

Our approaches are mainly based on cell and molecular biology techniques using Shear Stress Apparatus, live cell migration detection by Boyden’s chamber, live cell chamber and atomic force microscope (AFM) and gene and pharmacological delivery of NO.

Shear stress Apparatus

The cells used in this study are endothelial cells. Endothelial cells form a multi-functional lining of the intimal surface of blood vessels. This lining is continuously subjected to both steady and oscillatory fluid shear stresses, resulting from the flow of blood in the circulation.

Shear stress consists of two parts – Parallel plate flow chamber and Flow apparatus. The flow chamber, a polycarbonate plate, a rectangular Teflon gasket, and the glass slide with the attached endothelial cell monolayer. (We are thankful to Dr. Vijay Shah, Mayo Clinic, Rochester for providing the Parallel plate apparatus)

Parallel plate flow chamber assembly: the plate holder holds Polycarbonate plate (1 below), gasket and the glass slide with the cells attached on it, together (2-3 below). The medium enters through the entry port (A), through the slit (E), into the channel, and exits through the slit (F), and exit port (B). Valve (D) serves to remove the bubbles entered through the entry port (A).

A flow apparatus is capable of subjecting cultured cells to a wide range of steady shear stresses for long time periods. The apparatus consists of two reservoirs, situated one above the other, with a parallel-plate flow chamber positioned between them. The flow chamber is held in a metallic chamber to eradicate any leakage of the medium from it. Flow is driven through the chamber by the hydrostatic pressure head created by the vertical distance between the upper and lower reservoirs. The pressure head is maintained constant by continuous pumping of culture medium from the lower to upper reservoirs at rates in excess of that flowing through the chamber. The excess drains down the glass overflow manifold, which also serves to facilitate gas exchange with the medium. The reservoirs are made of glass, while the interconnecting tubing consists of Tylon except for the section through the roller pump, which is silicone. Silicone collars join the reservoirs to the manifold and tubing. The relatively inert and gas impermeable Tylon tubing prevents water and gas loss and minimizes absorption of cell metabolites.

The wall shear stress on the cell monolayer in the flow chamber may be calculated using the formula:

Table for different ranges of shear stress and corresponding height of the parallel flow plate:

Sl. No	Shear stress (dynes/ cm 2)	Height (in cms) at which the flow chamber placed (from the ground level)
1	1	110
2	5	98
3	10	75
4	15	57
5	20	39
6	25	18

References

Oren Traub; ; Bradford C. Berk. Laminar Shear Stress Mechanisms by Which Endothelial Cells Transduce an Atheroprotective Force. Arteriosclerosis, Thrombosis, and Vascular Biology. 1998.18:677-685.

Kristopher S Cunningham and Avrum I Gotlieb. The role of shear stress in the pathogenesis of atherosclerosis. Laboratory Investigation 2005. 85: 9–23.

Song Li, Ngan F. Huang, and Steven Hsu. Mechanotransduction in Endothelial Cell Migration. Journal of Cellular Biochemistry. 2005 96:1110–1126.

Aron B. Fisher, Shu Chien, Abdul I. Barakat, and Robert M. Nerem Endothelial cellular response to altered shear stress. Am J Physiol Lung Cell Mol Physiol. 2001. 281: L529-L533.

Mitchell.D.Botney. Role of Hemodynamics in Pulmonary Vascular Remodeling Implications for Primary Pulmonary Hypertension. Am J Respir Crit Care Med 1999 Vol 159. pp 361–364.

Vascular Biology Lab – Shear Stress

Physics Of Shear stress - Strain - Stress - Types of Stress

SS- Pathology - Implication - Cardiovascular blood flow - Atherosclerosis - Portal hypertension

PHYSICS OF SHEAR STRESS

STRAIN

STRESS

TYPES OF STRESS

SS- Pathology

Implication

Cardiovascular blood flow

Atherosclerosis

Portal hypertension

Implication

Cardiovascular blood flow

Atherosclerosis

Portal hypertension

Our Research Interest

Shear stress Apparatus

References

Research Labs working on Shear stress

Journals devoted to Shear Stress Research