Chapter 2 Physiologic Stress‐Mediated Signaling in the Endothelium

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Abstract

Although the vasculature was once thought to be a passive conduit for blood, it is now known that the endothelium is responsible for healthy vascular homeostasis and the progression of many cardiovascular‐related diseases. Because the endothelium lines blood vessels, it is subjected to the mechanical forces due to of blood flow. It is now well established that endothelial cells transduce these mechanical signals into chemical signals that are evident in the mechanoregulation of a number of signal transduction pathways and endothelial cell phenotype. Despite the significant volume of work in the field of endothelial cell mechanotransduction, the exact mechanism by which mechanical forces are sensed and transduced into chemical signals is not yet well established. In this chapter, we focus on the specific role of fluid shear stress, the frictional drag force caused by blood flow, and cyclic stretch caused by the pumping action of the heart, in regulating vascular homeostasis and vascular signaling. The regulation of flow‐mediated signaling in the endothelium is typically studied with well‐characterized in vitro flow and stretch devices. Here, we examine various platforms used to analyze flow‐mediated and stretch‐mediated signals and describe the method for the implementation of these techniques.

Introduction

The endothelial cell layer of the vasculature directly contacts the circulating blood, providing a dynamic barrier to the surrounding tissue. Because of its unique position in the body, it is exposed to three primary mechanical forces caused by blood flow: pressure caused by the hydrostatic force within the vessel, hoop stresses caused by the balance between the cell–cell contacts and vasomotion of the vessel, and the shear stresses caused by the friction of the blood flow against the vessel wall. In addition, the endothelium modulates a number of biologic processes within the vessel wall, including active regulation of vascular tone and blood pressure through the production of nitric oxide, control over the coagulation cascade and fibrinolytic processes through the production of prothrombotic and antithrombotic factors, regulation of vascular remodeling through the production of growth factors and vasoactive substances, and control over the inflammatory response by providing a platform by which leukocytes can home to inflamed tissue. As such, the endothelium mediates the progression of several pathologic conditions, including chronic inflammation, wound healing, and the development of various cardiovascular diseases including atherosclerosis.

In healthy blood vessels, endothelial cells are typically quiescent, presenting an antithrombotic nonadhesive surface to the passing blood (Garin and Berk, 2006, Pearson, 2000). However, on activation by cytokines, such as tumor necrosis factor (TNF‐α) or interleukin‐1β (IL‐1β), or other stimuli, endothelial cells upregulate a number of genes, including E‐selectin, intercellular adhesion molecule (ICAM)‐1, vascular cell adhesion molecule (VCAM)‐1, monocyte chemoattractant protein‐1 (MCP‐1), interleukin‐1 and 8, plasminogen activator inhibitor‐1 (PAI‐1), and tissue factor (TF) (Cybulsky, 1991, Diamond et al., 1989, Malek et al., 1999). (Fig. 2.1) In general, endothelial cell turnover increases, and the production of antithrombotic mediators decreases. Endothelial cell activation is required for a number of adaptive processes, including clotting and leukocyte adhesion during wound healing (Hajjar and Deora, 2000). However, chronic endothelial cell activation is linked to chronic inflammatory diseases, including atherosclerosis (Davignon and Ganz, 2004).

Chronic endothelial cell activation and increased incidence of atherosclerotic lesion formation is often detected in branch points or bifurcations within the vasculature (Gimbrone et al., 2000, Malek et al., 1999, World et al., 2006). Notably, the blood flow patterns, fluid shear stresses, and stretch patterns imposed on the endothelium are also strikingly different in these regions than those measured in the straight portions of the vessel (Chien, 2007). The nature and magnitude of blood flow, shear, and vessel wall stretch is largely determined by the shape and structure of the blood vessel and the cardiac cycle. The shear stresses found in most major human arteries have been found to be 2 to 20 dyn/cm2, with localized increases to 30 to 100 dyn/cm2 near branches and areas of sharp wall curvature (Dewey et al., 1981). Typically, in the straight portions of a blood vessel, the flow is laminar. In these regions, endothelial cells align and elongate parallel to the direction of flow (Fig. 2.2). This realignment corresponds with a streamlining of the cell that reduces resistance to flow and is speculated to mediate the subsequent signaling response. In contrast, flow within abrupt curvatures, such as occurs at bifurcations, is typically disturbed, exhibiting flow reversal, separation, and low velocity. As a result, the endothelial cells do not reorient like those located in the straight portions of the vessel. Because the cells do not align with the flow, their topology exposes them to greater shear stress gradients across the length of the cell, and these areas are also more prone to atherosclerosis (Barbee et al., 1994). For instance, within the carotid bifurcation, where atherosclerosis often develops, the flow separates, disrupting the laminar profile and producing disturbed streamlines (Ku et al., 1985). The lateral wall experiences areas of flow reversal and recirculation varying with the cardiac cycle, resulting in a time‐averaged shear stress close to zero. Because areas subjected to laminar shear stresses are generally free from plaque formation and lesions correlate with areas of disturbed flow, it is believed that laminar shear stresses impose an atheroprotective force on the vasculature and help maintain healthy vascular homeostasis (Berk et al., 2002, Traub and Berk, 1998). A similar general theme based on endothelial cell response to stretch has been advanced by Chien (Chien, 2007). However, the mechanism by which laminar flow and unidirectional stretch are detected and translated into an atheroprotective force by the endothelium remains unclear.

Significant evidence exists that laminar flow at physiologic shear stresses inhibits platelet aggregation and can enhance endothelial cell survival by preventing apoptosis (Dimmeler et al., 1996, Garin et al., 2007, Yoshizumi et al., 2003). Physiologic levels of cyclic stretch also seem to provide beneficial effects on endothelial barrier function (Fujiwara, 2003). In addition, significant evidence exists that disturbed flow that exerts time‐averaged low shear stresses on the endothelium induces endothelial expression of proapoptotic, proinflammatory, and procoagulant genes (Berk, 2008, Davies, 2007, Garcia‐Cardena and Gimbrone, 2006). However, the upstream events that initiate the signaling cascade in response to shear stress are still unclear.

Because many questions regarding how endothelial cells mechanotranduce fluid shear stress and stretch into intracellular responses, the atheroprotective effects of these forces and the subsequent flow‐mediated and stretch‐mediated signaling continue to be an active area of research. Although there have been significant advances in this field with animal models, in vivo work presents a number of challenges. In addition to the difficulties associated with isolating the effects of the mechanical signals caused by blood flow from the chemical humoral effectors, it is also difficult to visualize various force fields within the vasculature and to relate those stresses to specific endothelial cell phenotype in real time. Therefore, in vitro methods have been designed to mimic in vivo forces for the studies of flow‐mediated and stretch‐mediated signaling in the endothelium. Here, we will discuss several methods used to subject cells to flow, and we will also describe methods we have used to stretch cells to compare these mechanosignaling responses to the response elicited by flow.

Section snippets

Parallel Plate Flow System

The parallel plate flow chamber is one of the most commonly used platforms for subjecting monolayers of endothelial cells to uniform laminar shear stresses. Its advantages include the following (Kandlikar et al., 2005):

  • The fluid shear stress is relatively uniform within the chamber.

  • It can be used on a temperature‐controlled microscope stage, allowing for real‐time visualization of the cells and/or flow patterns within the chamber.

  • It is capable of applying a wide range of flow rates and fluid

Cone and Plate Flow System

The cone and plate flow system works almost identically to a cone and plate viscometer. Actually, many laboratories, including our own, have adapted cone and plate viscometers to subject uniform shear stress distribution within the fluid environment of a cell culture dish. A number of systems are commercially available, including those from Wells‐Brookfield, ThermoScientific, and Research Equipment Ltd., which can be adapted for use in cell culture. The cone and plate system consists of a cone

Cyclic Stretching of the Endothelium

In addition to fluid shear stress, endothelial cells are exposed to cyclic stretch caused by the pumping action of the heart. For large human arteries, the vessel wall expands circumferentially by 5 to 12% under the normal physiologic condition (Birukov et al., 2003, Nagel et al., 1999). Effects of mechanical stretch on endothelial cells and other cell types have been studied with various types of cell stretch apparatuses. These in vitro studies have shown that stretch activates specific

Acknowledgments

This work was supported by NHLBI grants HL 64839 and HL 77789 to B. C. B. and an NIH NRSA to C. R. K. (HL 84961).

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