Hemodynamics and Mechanobiology of Endothelium

The following sections are included:

• PREFACE

• CONTENTS

Atherosclerosis, the pathological formation of fibrous and lipid-rich plaques in large arteries, is a disease modulated in part by patterns of blood flow. Although all arteries tend to change in composition and become more fibrous with age, a process known as arteriosclerosis, atherosclerosis is a focal disease with unique pathology wherein the arterial wall thickens in distinct sites. Numerous studies have noted that atherosclerotic plaques form preferentially in specific locations, suggesting that the mechanics of flowing blood regulates such biological remodeling.¹ Wall shear stress resulting from the frictional force of blood dragging against its surrounding arteries has been shown in vitro and in vivo to modulate atherogenic processes.² Atherosclerosis is a multifactorial disease that is present in nearly all persons in the modern world, but biomechanics influences the location, progression, and resulting morbidity.

Emerging evidence shows that steady laminar flow is atheroprotective while disturbed flow creates an atheroprone environment in vivo. Chronic inflammation and reactive oxygen species production represent some of the pathogenic features of atherosclerosis formation, and it has become clear that steady laminar flow and disturbed flow or low shear stress have significant roles to modify these atherogenic events via regulating “mechanosignal transduction”. In this chapter, first we will discuss the possible pathological role of mechanosignal transduction in cardiovascular disease. Next the possible role of PECAM-1 as a mechanosensor and its regulatory mechanisms will be reviewed. Third, redox regulation induced by flow and its contribution to endothelial inflammation and apotosis will be summarized. Finally, we will discuss the interplay between cytokine-mediated inflammatory and laminar flow-mediated anti-inflammatory signaling in endothelial cells. The emphasis will be the regulatory mechanism between JNK and ERK5 and post-translational modification of ERK5 by SUMOylation. We believe that the clarification of these mechanosignaling pathways will lead us to understand the process of atherosclerosis formation in areas exposed to disturbed flow, especially in its initial phase of chronic inflammation.

The endothelial cell (EC) surface is coated with a glycocalyx (GCX) layer that is composed of a wide variety of membrane-bound macromolecules: sulfated proteoglycans, hyaluronic acid, sialic acids, glycoproteins, and plasma proteins. The GCX surface layer has been estimated to be from 20 nm to several microns thick in a static fluid environment, depending on cell/tissue preservation and microscopic visualization techniques that are reviewed in detail. Recently, there has been growing recognition that the GCX is a mechanotransducer and plays an important role in transmitting force to the cell's actin cytoskeleton and in initiating intracellular signaling. GCX thinning and shedding have been demonstrated to occur in diseases associated with mechanotransduction dysfunction. Recognizing the association of GCX degradation with disease, this chapter will provide an overview and synthesis of current knowledge concerning GCX molecular and physical structure and its function in endothelial mechanotransduction.

Laminar shear stress protects against atherogenesis and thrombosis by inducing anti-inflammatory and anti-coagulant gene expression in endothelial cells. Krüppel-like factors (KLFs) are zinc-finger type transcription factors that play important roles in modulating cellular function in a broad range of mammalian cell types. Several KLFs have been reported to be expressed in endothelial cells. Of these, KLF2 and KLF4 have been shown to be upregulated by laminar shear stress. Interestingly, the vasoprotective functions of shear stress overlap with described functions of KLF2, and accumulating evidence demonstrates that KLF2 mediates these favorable effects. This chapter will focus on the emerging role of Krüppel-like factors in endothelial cell biology with an emphasis on their role in laminar shear stress-mediated vasoprotection.

Fluid shear stress is a major determinant of endothelial cell shape, function, and gene transcription. Shear stress activates several signaling cascades in endothelial cells including the following: opening of K⁺ and CA⁺² channels,¹⁻³ activation of heterotrimeric G proteins,⁴ production of Nitric Oxide,⁵ tyrosine phosphorylation of proteins such as Shc, c-src, and focal adhesion kinase (FAK),^6,7 activation of MAP kinase pathway, protein kinase C (PKC) and jun C-terminal kinase (JNK),^8–10 release of reactive oxygen species (ROS),¹¹ and activation of important transcription factors such as c-fos, c-jun, c-myc and NF-kB.¹² The hallmark of the endothelial cell responses to fluid shear stress is the rearrangement of cytoskeleton and the elongation of microfilaments and microtubules in the direction of flow.^13–15 Shear stress can also modulate endothelial monolayer permeability of macromolecules¹⁶ and leukocytes from the blood into the underlying tissue, which is an early characteristic of atherosclerotic lesion development.¹⁷

A remarkable number of events stimulated by shear are downstream of small GTPases, and in particular Rho family GTPases. There are at least 150 small GTPases encoded by the human genome. The various subclasses of this superfamily, including Ras, Rho, Arf, Rab and Ran GTPases, have been implicated in almost all aspects on cell biology including proliferation, differentiation, cytoskeletal organization, vesicle trafficking, nucleocytoplasmic transport and gene expression.^18,19 All of the small GTPases function as binary ‘molecular switches’ that are active when bound to GTP and inactive when bound to GDP. The regulation of these states is tightly controlled by Guanine nucleotide Exchange Factors (GEFs) which activate the protein by exchanging a GDP for a GTP, and GTPase Activating Proteins (GAPs) which catalyze the hydrolysis of GTP to GDP to inactivate the protein.

The Rho family of small GTPases has been found to be critical in endothelial signaling pathways activated by shear stress, and the Rho subclass of GTPases will be the focus in this chapter. Three GTPases; namely, RhoA, Rac1 and Cdc42, are known to be crucial in regulating cell shape changes through the rearrangements of the cytoskeleton,²⁰ but they also have roles in endothelial adhesion, permeability and gene expression which are equally significant. This chapter will describe the well known roles for Rho family small GTPases in morphological and cytoskeletal rearrangements, and then discuss what is known about their roles in permeability and gene transcription in response to shear stress.

The greatest source of nitric oxide (NO) in the normal heart is the endothelial nitric oxide synthase (eNOS), which is localized in vascular endothelial cells (ECs). Endogenously synthesized NO has a variety of physiological functions.Its primary role is thought to be the control of vascular tone via its effect on the soluble guanylate cyclase enzyme of overlying smooth muscle cells. Cytochromec oxidase (CcO or complex IV), the terminal enzyme in the mitochondrial electron transport chain (ETC) is also a NO target. NO, via its effect on the mitochondrial ETC, can regulate cellular oxygen (O₂) consumption, superoxide radical production, and redox signaling. However, loss of the NO control over respiration can lead to increased formation of reactive O₂ and nitrogen species (ROS/RNS) and mitochondrial damage. Our group is particularly interested on the consequences of the NO interactions with endothelial mitochondria, because ECs under shear stress produce high levels of NO, and endothelial mitochondrial dysfunction is the hallmark of ischemia/reperfusion (I/RP) injury and is common in almost all vascular diseases. In this review, we discuss (1) the NO actions on mitochondria in general, and on mitochondria of cultured ECs exposed to shear stress in particular, and (2) the role of NO on the mitochondrial (dys)function of ECs and cardiomyocytes following I/RP. Using either ex vivo hearts or isolated mitochondria, it has been difficult to understand the relationship between NO, mitochondria and EC function. Cultured EC exposure to conditions of shear stress and O₂ tension (P_O2) that simulate the in vivo I/RP may provide additional insights.

In this chapter we highlight the contributions of genomics approaches to our understanding of endothelial phenotypic heterogeneity, particularly with respect to the influence of arterial hemodynamics and endothelial mechanobiology on normal vascular physiology and susceptibility to disease. Microarray approaches, experimental design and data analysis are reviewed so far as necessary to introduce the reader to the most important considerations, and a case study is presented to illustrate a typical workflow and analysis.

Atherosclerotic lesions preferentially locate at branch or bifurcation areas along the vessel wall, indicating that mechanical forces created by local flow patterns may exert different effect on the lesion development. Since the integrity of endothelium plays a role in the lesion formation, endothelial cell (EC) growth, proliferation and apoptosis that may be followed by endothelial progenitor cell (EPC) repair could be key issues. Recent evidence indicates that laminar shear stress can stimulate multi-potent stem or progenitor cells from different sources to differentiate toward EC lineage, which provides a promising cell source for cell-based therapeutic application in cardiovascular diseases. However, the underlying mechanism is far from well-understood. This chapter will give an overview of the effect of local flow pattern on EC differentiation, proliferation, survival and apoptosis and the possible underlying mechanism.

Current challenges in vascular medicine such as bypass grafting, stenting, and angioplasty have driven the field of vascular regenerative medicine and tissue engineering. Stem cells have shown promise as important components to these applications. Stem cells from multiple tissue sources have demonstrated the capability to differentiate into smooth muscle and endothelial cells in both laboratory and animal models. While early research focused on the biochemical mechanisms behind this phenomenon, more recent data demonstrated that mechanical stimulation can provide important signals to guide the differentiation of stem cells towards vascular phenotypes. The three mechanical stimuli relevant to the vascular system (stretch, shear, and pressure) have been applied in isolation or in combination to stem cells in two- and threedimensional systems. This chapter provides a review of such work to date, including the magnitude, frequency, and timing of mechanical stimuli used in guiding stem cell differentiation. Experiments utilizing concurrent biochemical stimulation are also discussed, highlighting the importance of a synergistic effort between the biochemical and biomechanical environments. Ultimately, a more complete understanding of these interactions will be necessary to yield important breakthroughs for the future of vascular regenerative medicine.

Recent advancements in congenital heart surgery have enabled children born with congenital cardiac anomalies to have longer and better quality lives. Most congenital heart operations require the use of either autologous or bioprosthetic materials to reconstruct the heart and great vessels; hence the need to develop an ideal vascular graft. The development of tissue-engineered vascular conduits and grafts hold great promise in further advancing the fields of pediatric cardiothoracic and vascular surgery. To date, implantation of such grafts in human patients has also been successful and has presented us with an exciting, rapidly advancing field.

The multifocal distribution of atherosclerotic lesions throughout the cardiovascular system correlates with arterial geometries that promote regions of disturbed flow. Disturbed blood flow develops due to the local boundary conditions and is more prevalent near branches and areas of small radii of curvature, arterial sites susceptible to lesion formation. Endothelial cells exposed to disturbed flow exhibit an atheroprone phenotype. Thus disturbed flow is a contributor to the development of arterial disease regardless of the physical length scale of the vessel. This should be considered when designing implantable endovascular devices that can change the local blood flow characteristics. Stenotic regions of arteries are mechanically opened through the deployment of commercial stents that introduce boundary conditions that also promote flow separation and flow reversal, conditions present at arterial sites where therosclerosis develops and where there is increased risk of thrombosis. This chapter considers how the merger of aerodynamic theory and local vessel hemodynamic conditions can serve as a design paradigm for a new generation of streamlined stent struts that inhibit the development of disturbed flow within the stented segment of a blood vessel.

Monocyte recruitment to inflamed endothelium is an early event in atherogenesis and occurs at focal regions where the average shear stress is on the order of one dyne/cm². Applying soft lithography to fabricate microfluidic channels that assemble above a living endothelial monolayer grown in culture, we have produced a vascular mimetic system that enables detailed studies of the influence of inflammation, hydrodynamics, and dietary lipid on the expression and function of vascular adhesion molecules.

To date, developing a translatable strategy to detect secondary flow in the athero-prone regions in vivo remains a challenge. To address this need, we have developed the MEMS thermal sensors to assess intravascular voltage (IVV) changes in the New Zealand White (NZW) rabbits on high fat/cholesterol diet. The MEMS thermal sensors were capable of sensing secondary flow as recorded by the significantly elevated time-averaged voltage values (V_ave) and the time-varying component (δV/δt) in the lesion-prone regions. These voltage profiles were distinct between the control and treated groups, corresponding to the histopathological findings for atheromas and foam cell infiltration. In this chapter, the heat transfer strategy will be introduced as a potential entry point to assess inflammatory responses, to demonstrate spatial and temporal variations in shear stress under a low Reynolds number flow in an arterial bifurcation model, and to detect sense secondary flow developed in the atheroprone regions of fat-fed NZW rabbits.

The following sections are included:

• INDEX